U.S. patent application number 14/777109 was filed with the patent office on 2016-02-04 for substituted heterocyclic compounds for treating or preventing viral infections.
The applicant listed for this patent is DANA-FARBER CANCER INSTITUTE, INC., PRESIDENT AND FELLOWS OF HARVARD COLLEGE. Invention is credited to Margaret J. Clark, Nathanael S. Gray, Chandrasekhar Miduturu, Priscilla Yang.
Application Number | 20160031826 14/777109 |
Document ID | / |
Family ID | 51581295 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160031826 |
Kind Code |
A1 |
Yang; Priscilla ; et
al. |
February 4, 2016 |
Substituted Heterocyclic Compounds for Treating or Preventing Viral
Infections
Abstract
Disclosed herein are pyrimidine compounds of formula I and
formula II and methods for treating or preventing a viral
infection, such as infections caused by dengue virus in a subject,
comprising administering to said subject an effective amount of a
pyrimidine compound of formula I or formula II.
Inventors: |
Yang; Priscilla; (Boston,
MA) ; Gray; Nathanael S.; (Boston, MA) ;
Miduturu; Chandrasekhar; (Cambridge, MA) ; Clark;
Margaret J.; (Zurich, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PRESIDENT AND FELLOWS OF HARVARD COLLEGE
DANA-FARBER CANCER INSTITUTE, INC. |
Cambridge
Boston |
MA
MA |
US
US |
|
|
Family ID: |
51581295 |
Appl. No.: |
14/777109 |
Filed: |
March 14, 2014 |
PCT Filed: |
March 14, 2014 |
PCT NO: |
PCT/US14/27654 |
371 Date: |
September 15, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61787956 |
Mar 15, 2013 |
|
|
|
Current U.S.
Class: |
514/235.8 ;
435/375; 514/256; 514/272; 514/275; 544/122; 544/321; 544/323;
544/328; 544/329 |
Current CPC
Class: |
C07D 239/47 20130101;
C07D 239/42 20130101; C07D 403/04 20130101; C07D 417/14 20130101;
C07D 413/14 20130101; C07D 239/48 20130101; C07D 239/69
20130101 |
International
Class: |
C07D 239/42 20060101
C07D239/42; C07D 403/04 20060101 C07D403/04; C07D 417/14 20060101
C07D417/14; C07D 239/48 20060101 C07D239/48; C07D 239/47 20060101
C07D239/47 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
Nos. AI057159-07 and AI095499-01 awarded by the National Institutes
of Health, National Institute of Allergy and Infectious Disease.
The government has certain rights in this invention.
Claims
1. A compound, or a pharmaceutically acceptable salt, solvate,
hydrate, prodrug, chemically-protected form, enantiomer or
stereoisomer thereof, wherein the compound is represented by
formula I or formula II ##STR00048## wherein, independently for
each occurrence, ##STR00049## is optionally substituted aryl or
optionally substituted heteroaryl; ##STR00050## is optionally
substituted aryl or optionally substituted heteroaryl; L.sup.1 is a
bond, --NR--, --O--, CR.sub.2--, --S--, --(CR.sub.2).sub.2--,
--C(O)--NR--, --NR--C(O)--, --C(O)O--, --O--C(O)--, --OCR.sub.2--,
--CR.sub.2O--, --NR--CR.sub.2--, or --CR.sub.2--NR--; L.sup.2 is a
bond, --NR--, --O--, CR.sub.2--, --S--, --(CR.sub.2).sub.2--,
--C(O)--NR--, --NR--C(O)--, --C(O)O--, --O--C(O)--, --OCR.sub.2--,
--CR.sub.2O--, --NR--CR.sub.2--, or --CR.sub.2--NR--; X is H or
halo; and R is H, alkyl, aryl, or aralkyl, provided the compound is
not ##STR00051##
2. The compound of claim 1, wherein ##STR00052## is substituted or
unsubstituted aryl.
3. The compound of claim 1, wherein ##STR00053## is substituted or
unsubstituted phenyl.
4. The compound of claim 1, wherein ##STR00054## is substituted or
unsubstituted aryl.
5. The compound of claim 1, wherein ##STR00055## is substituted or
unsubstituted phenyl.
6. The compound of claim 1, wherein ##STR00056## or ##STR00057## is
substituted with one or more substituents selected from the group
consisting of alkoxy, alkyl, --C(O)NR.sub.2, --C(O)OR, fluoroalkyl,
fluoroalkyloxy, aminoalkyl, hydroxyalkyl, halo, cyano, nitro, aryl,
heteroaryl, aralkyl, aryloxy, and heteroaryloxy.
7. The compound of claim 1, wherein ##STR00058## or ##STR00059## is
substituted with one or more substituents selected from the group
consisting of alkoxy, fluoroalkyl, fluoroalkyloxy, aminoalkyl,
hydroxyalkyl, halo, cyano, and nitro.
8. The compound of claim 1, wherein ##STR00060## or ##STR00061## is
substituted with one or more substituents selected from the group
consisting of --OCF.sub.3 and --CF.sub.3.
9. The compound of claim 1, wherein L.sup.1 is --NR-- or --O--.
10-12. (canceled)
13. The compound of claim 1, wherein L.sup.2 is --NR-- or
--O--.
14-15. (canceled)
16. The compound of claim 1, wherein the compound is selected from
the group consisting of ##STR00062## ##STR00063## ##STR00064##
17. (canceled)
18. A method of inhibiting entry of a virus into a host cell
comprising contacting the host cell with an effective amount of a
compound of claim 1 or ##STR00065##
19-20. (canceled)
21. The method of claim 18, wherein the virus is of family
Flaviviridae.
22-23. (canceled)
24. The method of claim 18, wherein the virus is dengue virus
(DENV).
25. A method of treating or preventing a viral infection in a
subject comprising administering to the subject an effective amount
of a compound of claim 1 or ##STR00066##
26-27. (canceled)
28. The method of claim 25, wherein the viral infection is a result
of a virus of family Flaviviridae.
29-30. (canceled)
31. The method of claim 25, wherein the viral infection is a result
of dengue virus (DENV).
32. The method of claim 25, wherein the viral infection is selected
from the group consisting of: Dengue fever, Japanese encephalitis,
Kyasanur Forest disease, Murray Valley encephalitis, St. Louis
encephalitis, Tick-borne encephalitis, West Nile encephalitis,
Yellow fever, and Hepatitis C.
33. The method of claim 18, wherein the wherein the compound is
selected from the group consisting of ##STR00067## ##STR00068##
34. The method of claim 25, wherein the wherein the compound is
selected from the group consisting of ##STR00069## ##STR00070##
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application Ser. No. 61/787,956, filed Mar. 15,
2013; the contents of which are hereby incorporated by
reference.
BACKGROUND
[0003] The flavivirus family includes several clinically important
animal viruses, including Dengue, West Nile, Japanese encephalitis,
yellow fever, and tick-borne encephalitis viruses. Dengue is one of
the most serious infectious diseases globally. There are about 300
million cases every year, with over 500,000 cases of potentially
fatal Dengue hemorrhagic fever. Dengue virus (DENV) puts nearly 2.5
billion people at risk of infection in tropical and subtropical
countries. Similarly, West Nile virus (WNV) has caused thousands of
human infections in North America, besides infecting people on
other continents. WNV infection can lead to serious illnesses in
humans, resulting in encephalitis and death. Neither a prophylactic
vaccine nor antiviral therapies are available for both WNV and
DENV. The development of either a vaccine or an antiviral drug
requires detailed knowledge of the viral life cycle.
[0004] The spherical, approximately 50 nm diameter dengue virion
contains an inner nucleocapsid made up of the plus-sense RNA genome
and multiple copies of the viral core protein and is sheathed in a
lipid bilayer derived from the host cell. The virion membrane is
coated with 180 copies of the envelope (E) protein that form 90
homodimers arranged in a tight herringbone structure. Following
attachment to the cell surface, the dengue virion is internalized
via a clathrin-dependent process. The viral nucleocapsid escapes
from the endosomal compartment via a pore created upon fusion of
the viral and endosomal membranes. This process of viral fusion is
catalyzed by the E protein and is triggered by acidic pH. The viral
genome is translated to produce a single polyprotein that is
post-translationally processed by cellular and viral proteases to
produce the ten DENV proteins. Replication of the viral genome is
catalyzed by the viral RNA-dependent RNA polymerase, NS5, and
occurs in membrane-associated complexes in the perinuclear region.
Upon encapsidation of the viral genomic RNA by the core protein,
the nucleocapsids bud into the endoplasmic reticulum lumen, a
process that leads to their acquisition of a lipid membrane and
association with 180 heterodimers of the viral E and prM proteins
organized as quasi-trimers arranged perpendicularly to the virion
surface. prM functions as a chaperone protein to prevent premature
triggering of the E protein on immature viral particles within the
acidic environment of the secretory pathway. Mature viral particles
are produced upon cleavage of prM by furin and rearrangement of E
into homodimers during exocytosis. The DENV infectious cycle is
known to occur on the timescale of several hours, with the release
of progeny viral particles commencing at 12 to 24 hours following
infection, depending upon the virus strain and cell-type.
[0005] Based on its tertiary structure, the DENV E protein is a
class II viral fusion protein. Two transmembrane domains anchor the
E protein in the viral membrane and are linked to three globular
domains (domains I, II, and III) via a short "stem" region and
membrane proximal helix-loop-helix. Domain I is a .beta.-barrel
forming the core of the protein monomer. The immunoglobulin-like
domain III acts as the putative receptor-binding domain and is the
major site of neutralizing antibody epitopes. The fusion loop
located at the tip of the "finger-like" domain II contacts domain
III of the dimer partner. The large-scale structural changes
triggered by the acidification of the endosomal compartment
catalyze fusion of the viral and target membranes.
[0006] Viruses can be interrogated using chemical tools; for
example, small molecules or RNAi may be used to identify host
factors or pathways integral for viral replication or viral entry.
Specifically, host proteins and enzymes that may be important for
viral replication may be probed by measuring the effect on yield of
viral particles in the presence of known inhibitors of a specific
protein or enzyme. In addition, small molecules may inhibit viral
entry by interacting with viral proteins, such as envelope protein
E.
[0007] In general, there exists a need for antiviral therapies that
are not detrimental to host cell viability.
SUMMARY
[0008] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
chemically-protected form, enantiomer or stereoisomer thereof,
wherein the compound is represented by formula I or formula II
##STR00001##
wherein, independently for each occurrence,
##STR00002##
is optionally substituted aryl or optionally substituted
heteroaryl;
##STR00003##
is optionally substituted aryl or optionally substituted
heteroaryl;
[0009] L.sup.1 is a bond, --NR--, --O--, CR.sub.2--, --S--,
--(CR.sub.2).sub.2--, --C(O)--NR--, --NR--C(O)--, --C(O)O--,
--O--C(O)--, --OCR.sub.2--, --CR.sub.2O--, --NR--CR.sub.2--, or
--CR.sub.2--NR--;
[0010] L.sup.2 is a bond, --NR--, --O--, CR.sub.2--, --S--,
--(CR.sub.2).sub.2--, --C(O)--NR--, --NR--C(O)--, --C(O)O--,
--O--C(O)--, --OCR.sub.2--, --CR.sub.2O--, --NR--CR.sub.2--, or
--CR.sub.2--NR--;
[0011] X is H or halo; and
[0012] R is H, alkyl, aryl, or aralkyl,
[0013] provided the compound is not
##STR00004##
[0014] In certain embodiments, the invention relates to a method of
inhibiting entry of a virus into a host cell comprising contacting
the host cell with an effective amount of any one of the
aforementioned compounds.
[0015] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the virus is dengue virus
(DENV).
[0016] In certain embodiments, the invention relates to a method of
treating or preventing a viral infection in a subject comprising
administering to the subject, (e.g., a subject in need thereof), an
effective amount of any one of the aforementioned compounds.
[0017] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the viral infection is a result
of dengue virus (DENV).
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 depicts a screen of kinase inhibitors with anti-DENV
activity. Several Abl kinase inhibitors exhibit activity against
dengue virus.
[0019] FIG. 2 depicts that DENV replication is reduced in cell
lines lacking Abl kinase activity (left set of data=MOI 10; right
set of data=MOI 1).
[0020] FIG. 3 depicts that DENV replication is reduced in cell
lines lacking Abl kinase activity.
[0021] FIG. 4 depicts the reduction in DENV viral titer by
imatinib.
[0022] FIG. 5 depicts the reduction in DENV viral titer by
GNF2.
[0023] FIG. 6 depicts the results from Example 3 (DMSO=left bar;
GNF2=middle bar; and imatinib=right bar).
[0024] FIGS. 7-9 depicts GNF2 and various GNF2 analogs. The
activities of these compounds indicate that kinase- and
virion-mediated activities are separate.
[0025] FIG. 10 depicts an assay investigating the pulldown of
purified virus with biotinylated GNF2.
[0026] FIG. 11 depicts a schematic showing the experimental setup
for a fluorescence polarization assay to measure binding of GNF2 to
DENV E protein. Binding of GNF2-FITC (GNF2-fluorescein
isothiocyanate) by E dimer decreases rate of tumbling and increases
polarization of the emitted fluorescence.
[0027] FIG. 12 depicts the detection of binding of GNF2-FITC to
recombinant DENV sE prefusion dimer by fluorescence
polarization.
[0028] FIG. 13 depicts the use of fluorescent microscopy to track
GNF2-CY5 during DENV entry. GNF2-CY5 retains its antiviral activity
as compared to GNF2.
[0029] FIG. 14 depicts a schematic representation of the
interaction between DENV and a liposome.
[0030] FIG. 15 depicts an assay of DENV fusion in the presence or
absence of a compound of the invention.
[0031] FIG. 16 depicts an assay indicating that GNF2 does not block
association of DENV with liposomes.
[0032] FIGS. 17-21 tabulate the IC.sub.90 and LD.sub.90 values for
various compounds of the invention. In particular, the tables
demonstrate that compounds of the invention have activity against
multiple serotypes of DENV. Certain compounds of the invention
exhibit pan-serotype activity.
[0033] FIGS. 22-29 depict the IC.sub.90 values associated with
various compounds of the invention.
[0034] FIG. 30 depicts that GNF-2 has additive effects on DENV
titer. All virus and cell treatments were done at EC.sub.90 values
determined empirically. Virus treatment was carried out for 45
minutes at 37.degree. C. prior to initial cell infection (MOI 1).
Treatment of BHK-21 cells was begun immediately after the initial
one-hour infection. Each bar represents the mean of three
replicates with error bars showing standard deviation. * indicates
p value<0.01.
[0035] FIG. 31 tabulates the IC.sub.50 for various
4,6-disubstituted compounds of the invention. Virus was
preincubated with 10 or 100 micromolar compound. Excess compound
was removed by size exclusion then used to infect cells at MOI 1.
Viral yield determined at 24 h post-infection. Log 10 unit decrease
indicated in columns on the right.
[0036] FIG. 32 depicts various 4,6-disubstituted compounds of the
invention.
[0037] FIGS. 33 and 34 depict fluorescence polarization data
showing that the 2,4-disubstituted compounds compete with GNF2-FITC
for binding to E. The competition is complete by 2 h (FIG. 33) for
the 4,6-disubstituted derivatives (1-100-1, 1-97-3) but takes until
12 h (FIG. 34) for the 2,4-disubstituted compounds (2-12-3,
2-21-2).
[0038] FIGS. 35 and 36 depict compounds of the invention comprising
biotin, FITC, or BoDIPY functionality.
[0039] FIG. 37 depicts compounds of the invention comprising a
solubility-enhancing group.
[0040] FIG. 38 tabulates the EC90, IC50, and KD concentrations for
various compounds of the invention (see FIGS. 35-37). All EC90 and
KD values were measured against dengue serotype 2 (DENV2).
[0041] FIG. 39 depicts viral titer as a function of number of
passages, which was used in the selection of a mutant resistant to
various compounds.
[0042] FIG. 40 depicts the confirmation that M196V mutation in
DENV2 E confers decreased sensitivity to 7-148-6.
[0043] FIG. 41 depicts decreased sensitivity of DENV2 with an M196V
E mutation to various compounds.
[0044] FIG. 42 depicts the KD of DENV2 sE.sub.2(wt) to
GNF2-biotin.
[0045] FIG. 43 depicts the KD of DENV2 sE.sub.2(M196V) to
GNF2-biotin.
DETAILED DESCRIPTION
Overview
[0046] In certain embodiment, the invention relates to the effect
of small molecules (compounds or active agents) on viral
replication. In certain embodiments, the small molecules interact
with host cell factors, such as enzymes, and so perturb viral
replication. In certain embodiments, the host cell factor is a
kinase. Kinases play a central role in intracellular signal
transduction and regulate many cellular processes, including cell
division, apoptosis, immune activation, trafficking, and cell
survival. In certain embodiments, the host cell factor is a
tyrosine kinase.
[0047] In certain embodiments, the compound is an inhibitor of
intracellular Abl kinase. In certain embodiments, the compound
blocks viral entry independent of its Abl kinase activity. In
certain embodiments, the compound binds directly to a protein
associated with the virion. In certain embodiments, the compound
binds directly to a virion envelope protein. In certain
embodiments, the compound blocks DENV fusion at a step after
insertion of the fusion loops.
[0048] GNF-2, an allosteric inhibitor of the Bcr-Abl kinase and
other Abl kinases, was identified as an inhibitor of the infectious
cycle of DENV in cell culture. In certain embodiments, the
invention relates to efforts to elucidate the mechanism by which
GNF-2 inhibits DENV2. In certain embodiments, the invention relates
to the discovery that GNF-2's inhibition of DENV is mediated by two
distinct mechanisms, one mediated by Abl kinases and one that is
independent of Abl kinases and instead mediated by the DENV E
protein on the virion surface. In dose-response and order of
addition experiments, GNF-2 inhibited DENV infectivity when
pre-incubated with the viral inoculum; however, this inhibition
could not be entirely recapitulated with imatinib, a comparably
potent but structurally distinct inhibitor of Abl kinases.
Employing a chemical biology approach, we synthesized derivatives
of GNF-2 conjugated to biotin and FITC and then demonstrated the
interaction of these compounds with purified dengue virions and
recombinant proteins corresponding to the soluble prefusion dimer
(sE.sub.2), respectively. We furthermore performed a focused
medicinal chemistry study to elucidate differences in the
structure-activity relationships underlying GNF-2's kinase
inhibitory activity versus those responsible for its inhibition of
DENV infectivity, leading to the identification of compound 2-12-2,
a 2,4-disubstituted pyrimidine that lacks cellular activity against
Abl kinases but whose inhibition of DENV infectivity is improved
relative to GNF-2. In certain embodiments, compound 2-12-2 blocks
fusion of dengue virions with model liposomes in vitro, suggesting
that its interaction with E prevents membrane fusion during DENV
entry.
ABL Tyrosine Kinases and Abl Kinase Inhibitors
[0049] ABL-family proteins comprise one of the best conserved
brandies of the tyrosine kinases. Each ABL protein contains an
SH3-SH2-TK (Src homology 3-Src homology 2-tyrosine kinase) domain
cassette, which confers autoregulated kinase activity and is common
among nonreceptor tyrosine kinases. This cassette is coupled to an
actin-binding and -bundling domain, which makes ABL proteins
capable of connecting phosphoregulation with actin-filament
reorganization. Two vertebrate paralogs, ABL1 and ABL2, have
evolved to perform specialized functions. ABU includes nuclear
localization signals and a DNA binding domain through which it
mediates DNA damage-repair functions, whereas ABL2 has additional
binding capacity for actin and for microtubules to enhance its
cytoskeletal remodeling functions. Several types of
posttranslational modifications control ABL catalytic activity,
subcellular localization, and stability, with consequences for both
cytoplasmic and nuclear ABL functions. Binding partners provide
additional regulation of ABL catalytic activity, substrate
specificity, and downstream signaling.
[0050] Bcr-Abl tyrosine-kinase inhibitors (TKI) are the first-line
therapy for most patients with chronic myelogenous leukemia (CML).
In more than 90% cases CML is caused by chromosomal abnormality
resulting in the formation of a so-called Philadelphia chromosome.
Compounds have been developed that selectively inhibit this
tyrosine kinase. Before the U.S. Food and Drug Administration (FDA)
approval of imatinib in 2001 no drugs were used that changed the
natural progression of CML, only cytotoxic drugs such as busulfan,
hydroxyurea or interferon-alpha (rIFN-.alpha.). Most of the drugs
are adenosine triphosphate (ATP)-competitive inhibitors.
Small Molecule Inhibitors of DENV Entry
[0051] In certain embodiments, the invention relates to the
identification of an unexpected, previously unknown viral target
for GNF-2, known to be a small molecule inhibitor of intracellular
Abl kinases. GNF-2 reduces DENV infectivity as measured in yield
reduction assays either when present on the cells after initial
infection or when the compound is pre-incubated with virus
inoculum. This latter inhibition appears to be Abl-independent and
target the dengue virion itself Using GNF-2 as a scaffold, we were
able to identify several small molecule inhibitors of DENV entry
that were inactive against Abl kinases.
[0052] GNF-2's anti-DENV activity during entry is not as potent as
several other published DENV entry inhibitors, but it is unique
among these entry inhibitors due to the fact that it also inhibits
DENV at a later stage of the viral life cycle, most likely via its
inhibition of cellular Abl kinases. GNF-2's inhibition of DENV via
two separate targets via two independent mechanisms of action leads
to the possible concept of dual-action viral inhibitors. The
concept of using multi-targeted compounds to achieve a maximal
therapeutic index is gaining traction in other areas of
biomedicine, notably oncology. This concept has been tentatively
explored previously in other viruses. One HIV study identified a
peptide that inhibits glycoprotein gp120 interactions with both of
its cell protein ligands, while another study discovered a small
molecule that inhibits two separate steps of the HIV integration
process. For rhinovirus, an inhibitor was identified that prevented
activity of two separate viral proteases. However, our results for
GNF-2 reveal a molecule that inhibits not at two points during one
step of the viral life cycle, but rather acts at two separate
points in the viral life cycle and is likely doing so via two
separate targets. This concept is not without precedent; a
dendrimer was identified that inhibited both herpes simplex virus
(HSV) and late stages of viral replication, although potential
target(s) of the molecule were not explored. We propose that it may
be possible to use rational design to identify molecules that can
inhibit viruses via two separate targets at separate points in the
viral life cycle. The targets could be viral, cellular, or both, as
we believe is the case for GNF-2. The challenging part of this
approach would be to balance optimization of inhibitor activity
between the two targets. While such dual-action inhibitors would be
attractive due to their potentially higher barriers to viral
resistance, this proposition remains to be experimentally
explored.
[0053] The order of addition and the MOI titration experiments, as
well as the fact that 2-12-2 inhibits DENV fusion in vitro, a
process mediated by E, strongly suggest that small molecule 2-12-2,
identified in the SAR study, targets the virion or dengue E
protein. It is possible, although unlikely, that GNF-2 and 2-12-2
interact non-specifically with the lipid envelope of the virus. In
addition, the location of binding on the E protein remains unknown
for GNF-2 and 2-12-2; it is possible that binding sites for these
two molecules may differ. We hypothesize that GNF-2 also inhibits
DENV infectivity at the step of viral fusion, but due to the
parental compound's lower potency, we were not able to test it in
our in vitro assays. Thus, it is possible that GNF-2 may block a
different step of DENV entry. Regardless of the step GNF-2
inhibits, however, it proved valuable as a scaffold in our SAR
study, and led to the identification of several molecules that were
up to five times more potent in cellular assays.
[0054] One potential binding location on the E protein for GNF-2 is
the "hinge region" located between domains I and II. This site has
previously been shown to be a ligand binding site, as
crystallization of recombinant E revealed a single molecule of
beta-octoglucoside (BOG) in the pocket formed by domains I and II.
Several other small molecule inhibitors of DENV entry have also
been hypothesized to bind in this area. However, binding in this
region was originally predicted to inhibit the extension of domain
II of E away from the viral membrane and hence block the insertion
of the fusion loops into a target membrane. Interestingly, the
M196V resistance mutation, described herein, is located at the base
of this pocket. This mutation reduces the affinity of soluble
prefusion E dimer for a biotinylated version of GNF2 by 30-fold
(K.sub.D wildtype around 1 .mu.M, K.sub.D mutant around 30 .mu.M).
This mutation also decreases the sensitivity of dengue serotype 2
reporter viruses. These observations are consistent with the idea
that GNF2, 2-12-2, etc., may bind in the BOG pocket.
[0055] A previous study identified two other regions of the E
protein that were predicted to be potential small molecule binding
locations that prevent conformational changes of E that occur after
insertion of the fusion (Yennamalli et al., 2009), so it is
possible that our compounds could bind in either of these
additional locations.
[0056] The work presented here reveals that GNF-2, a previously
identified Abl kinase inhibitor, has an Abl-independent inhibitory
effect on DENV entry, and this effect is likely mediated through
interactions with the dengue virion itself Using GNF-2 as a
scaffold, we were able to identify several disubstituted
pyrimidines that lower DENV yield by one log at single-digit
micromolar concentrations when pre-incubated with virus inoculum.
Further exploration of GNF-2 as a scaffold may identify even more
potent DENV entry inhibitors. In addition, this study raises the
possibility of rationally designing dual-target small molecule
inhibitors of virus infection.
DEFINITIONS
[0057] In order for the invention to be more readily understood,
certain terms and phrases are defined below and throughout the
specification.
[0058] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0059] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0060] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e., "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0061] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0062] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0063] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
[0064] The definition of each expression, e.g., alkyl, m, n, and
the like, when it occurs more than once in any structure, is
intended to be independent of its definition elsewhere in the same
structure.
[0065] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., a compound which does not spontaneously undergo
transformation such as by rearrangement, cyclization, elimination,
or other reaction.
[0066] The term "substituted" is also contemplated to include all
permissible substituents of organic compounds. In a broad aspect,
the permissible substituents include acyclic and cyclic, branched
and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic substituents of organic compounds. Illustrative
substituents include, for example, those described herein below.
The permissible substituents may be one or more and the same or
different for appropriate organic compounds. For purposes of this
invention, the heteroatoms such as nitrogen may have hydrogen
substituents and/or any permissible substituents of organic
compounds described herein which satisfy the valences of the
heteroatoms. This invention is not intended to be limited in any
manner by the permissible substituents of organic compounds.
[0067] The term "lower" when appended to any of the groups listed
below indicates that the group contains less than seven carbons
(i.e. six carbons or less). For example "lower alkyl" refers to an
alkyl group containing 1-6 carbons, and "lower alkenyl" refers to
an alkenyl group containing 2-6 carbons.
[0068] The term "saturated," as used herein, pertains to compounds
and/or groups which do not have any carbon-carbon double bonds or
carbon-carbon triple bonds.
[0069] The term "unsaturated," as used herein, pertains to
compounds and/or groups which have at least one carbon-carbon
double bond or carbon-carbon triple bond.
[0070] The term "aliphatic," as used herein, pertains to compounds
and/or groups which are linear or branched, but not cyclic (also
known as "acyclic" or "open-chain" groups).
[0071] The term "cyclic," as used herein, pertains to compounds
and/or groups which have one ring, or two or more rings (e.g.,
spiro, fused, bridged).
[0072] The term "aromatic" refers to a planar or polycyclic
structure characterized by a cyclically conjugated molecular moiety
containing 4n+2 electrons, wherein n is the absolute value of an
integer. Aromatic molecules containing fused, or joined, rings also
are referred to as bicyclic aromatic rings. For example, bicyclic
aromatic rings containing heteroatoms in a hydrocarbon ring
structure are referred to as bicyclic heteroaryl rings.
[0073] The term "hydrocarbon" as used herein refers to an organic
compound consisting entirely of hydrogen and carbon.
[0074] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover.
[0075] The term "heteroatom" as used herein is art-recognized and
refers to an atom of any element other than carbon or hydrogen.
Illustrative heteroatoms include boron, nitrogen, oxygen,
phosphorus, sulfur and selenium.
[0076] The term "alkyl" means an aliphatic or cyclic hydrocarbon
radical containing from 1 to 12 carbon atoms. Representative
examples of alkyl include, but are not limited to, methyl, ethyl,
n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,
n-pentyl, isopentyl, neopentyl, n-hexyl, 2-methylcyclopentyl, and
1-cyclohexylethyl.
[0077] The term "substituted alkyl" means an aliphatic or cyclic
hydrocarbon radical containing from 1 to 12 carbon atoms,
substituted with 1, 2, 3, 4, or 5 substituents independently
selected from the group consisting of alkyl, alkenyl, alkynyl,
halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkenyloxy,
alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy,
fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio,
fluoroalkylthio, alkenylthio, alkynylthio, sulfonic acid,
alkylsulfonyl, haloalkylsulfonyl, fluoroalkylsulfonyl,
alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl,
haloalkoxysulfonyl, fluoroalkoxysulfonyl, alkenyloxysulfonyl,
alkynyloxysulfonyl, aminosulfonyl, sulfinic acid, alkylsulfinyl,
haloalkylsulfinyl, fluoroalkylsulfinyl, alkenylsulfinyl,
alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl,
fluoroalkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny,
aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl,
fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy,
alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl,
alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, halo
alkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy,
alkynylcarbonyloxy, alkylsulfonyloxy, halo alkylsulfonyloxy,
fluoroalkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy,
haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy,
alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,
halo alkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,
alkynylsulfinyloxy, alkoxysulfinyloxy, halo alkoxysulfinyloxy,
fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy,
alkynyloxysulfinyloxy, aminosulfinyloxy, amino, amido,
aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl,
phosphoryl, silyl and silyloxy.
[0078] The term "alkylene" is art-recognized, and as used herein
pertains to a bidentate moiety obtained by removing two hydrogen
atoms of an alkyl group, as defined above.
[0079] The term "alkenyl" as used herein means a straight or
branched chain hydrocarbon containing from 2 to 10 carbons and
containing at least one carbon-carbon double bond formed by the
removal of two hydrogens. Representative examples of alkenyl
include, but are not limited to, ethenyl, 2-propenyl,
2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl,
2-methyl-1-heptenyl, and 3-decenyl.
[0080] The term "alkynyl" as used herein means a straight or
branched chain hydrocarbon group containing from 2 to 10 carbon
atoms and containing at least one carbon-carbon triple bond.
Representative examples of alkynyl include, but are not limited, to
acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and
1-butynyl.
[0081] The term "carbocyclyl" as used herein means monocyclic or
multicyclic (e.g., bicyclic, tricyclic, etc.) hydrocarbons
containing from 3 to 12 carbon atoms that is completely saturated
or has one or more unsaturated bonds, and for the avoidance of
doubt, the degree of unsaturation does not result in an aromatic
ring system (e.g. phenyl). Examples of carbocyclyl groups include
1-cyclopropyl, 1-cyclobutyl, 2-cyclopentyl, 1-cyclopentenyl,
3-cyclohexyl, 1-cyclohexenyl and 2-cyclopentenylmethyl.
[0082] The term "heterocyclyl", as used herein include
non-aromatic, ring systems, including, but not limited to,
monocyclic, bicyclic (e.g. fused and spirocyclic) and tricyclic
rings, which can be completely saturated or which can contain one
or more units of unsaturation, for the avoidance of doubt, the
degree of unsaturation does not result in an aromatic ring system,
and have 3 to 12 atoms including at least one heteroatom, such as
nitrogen, oxygen, or sulfur. For purposes of exemplification, which
should not be construed as limiting the scope of this invention,
the following are examples of heterocyclic rings: azepines,
azetidinyl, morpholinyl, oxopiperidinyl, oxopyrrolidinyl,
piperazinyl, piperidinyl, pyrrolidinyl, quinicludinyl,
thiomorpholinyl, tetrahydropyranyl and tetrahydrofuranyl. The
heterocyclyl groups of the invention are substituted with 0, 1, 2,
3, 4 or 5 substituents independently selected from the group
consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl,
fluoroalkyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy,
carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy,
sulfhydryl, alkylthio, haloalkylthio, fluoroalkylthio, alkenylthio,
alkynylthio, sulfonic acid, alkylsulfonyl, haloalkylsulfonyl,
fluoroalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl,
alkoxysulfonyl, haloalkoxysulfonyl, fluoroalkoxysulfonyl,
alkenyloxysulfonyl, alkynyloxysulfonyl, aminosulfonyl, sulfinic
acid, alkylsulfinyl, haloalkylsulfinyl, fluoroalkylsulfinyl,
alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl, halo
alkoxysulfinyl, fluoroalkoxysulfinyl, alkenyloxysulfinyl,
alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,
haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,
alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,
fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,
alkylcarbonyloxy, halo alkylcarbonyloxy, fluoroalkylcarbonyloxy,
alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, halo
alkylsulfonyloxy, fluoroalkylsulfonyloxy, alkenylsulfonyloxy,
alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy,
alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,
halo alkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,
alkynylsulfinyloxy, alkoxysulfinyloxy, halo alkoxysulfinyloxy,
fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy,
alkynyloxysulfinyloxy, aminosulfinyloxy, amino, amido,
aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl,
phosphoryl, silyl, silyloxy, and any of said substituents bound to
the heterocyclyl group through an alkylene moiety (e.g.
methylene).
[0083] The term "N-heterocyclyl" as used herein is a subset of
heterocyclyl, as defined herein, which have at least one nitrogen
atom through which the N-heterocyclyl moiety is bound to the parent
moiety. Representative examples include pyrrolidin-1-yl,
piperidin-1-yl, piperazin-1-yl, hexahydropyrimidin-1-yl,
morpholin-1-yl, 1,3-oxazinan-3-yl and 6-azaspiro[2.5]oct-6-yl. As
with the heterocyclyl groups, the N-heterocyclyl groups of the
invention are substituted with 0, 1, 2, 3, 4 or 5 substituents
independently selected from the group consisting of alkyl, alkenyl,
alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkenyloxy,
alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy,
fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio,
fluoroalkylthio, alkenylthio, alkynylthio, sulfonic acid,
alkylsulfonyl, haloalkylsulfonyl, fluoroalkylsulfonyl,
alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl,
haloalkoxysulfonyl, fluoroalkoxysulfonyl, alkenyloxysulfonyl,
alkynyloxysulfonyl, aminosulfonyl, sulfinic acid, alkylsulfinyl,
haloalkylsulfinyl, fluoroalkylsulfinyl, alkenylsulfinyl,
alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl,
fluoroalkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny,
aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl,
fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy,
alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl,
alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, halo
alkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy,
alkynylcarbonyloxy, alkylsulfonyloxy, halo alkylsulfonyloxy,
fluoroalkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy,
haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy,
alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,
halo alkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,
alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy,
fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy,
alkynyloxysulfinyloxy, aminosulfinyloxy, amino, amido,
aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl,
phosphoryl, silyl, silyloxy, and any of said substituents bound to
the N-heterocyclyl group through an alkylene moiety (e.g.
methylene).
[0084] The term "aryl," as used herein means a phenyl group,
naphthyl or anthracenyl group. The aryl groups of the invention can
be optionally substituted with 1, 2, 3, 4 or 5 substituents
independently selected from the group consisting of alkyl, alkenyl,
alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkenyloxy,
alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy,
fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio,
fluoroalkylthio, alkenylthio, alkynylthio, sulfonic acid,
alkylsulfonyl, haloalkylsulfonyl, fluoroalkylsulfonyl,
alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl,
haloalkoxysulfonyl, fluoroalkoxysulfonyl, alkenyloxysulfonyl,
alkynyloxysulfonyl, aminosulfonyl, sulfinic acid, alkylsulfinyl,
haloalkylsulfinyl, fluoroalkylsulfinyl, alkenylsulfinyl,
alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl,
fluoroalkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny,
aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl,
fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy,
alkoxycarbonyl, halo alkoxycarbonyl, fluoroalkoxycarbonyl,
alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy,
haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy,
alkynylcarbonyloxy, alkylsulfonyloxy, halo alkylsulfonyloxy,
fluoroalkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy,
haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy,
alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,
halo alkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,
alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy,
fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy,
alkynyloxysulfinyloxy, aminosulfinyloxy, amino, amido,
aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl,
phosphoryl, silyl, silyloxy, and any of said substituents bound to
the heterocyclyl group through an alkylene moiety (e.g.
methylene).
[0085] The term "arylene," is art-recognized, and as used herein
pertains to a bidentate moiety obtained by removing two hydrogen
atoms of an aryl ring, as defined above.
[0086] The term "arylalkyl" or "aralkyl" as used herein means an
aryl group, as defined herein, appended to the parent molecular
moiety through an alkyl group, as defined herein. Representative
examples of aralkyl include, but are not limited to, benzyl,
2-phenylethyl, 3-phenylpropyl, and 2-naphth-2-ylethyl.
[0087] The term "biaryl," as used herein means an aryl-substituted
aryl, an aryl-substituted heteroaryl, a heteroaryl-substituted aryl
or a heteroaryl-substituted heteroaryl, wherein aryl and heteroaryl
are as defined herein. Representative examples include
4-(phenyl)phenyl and 4-(4-fluorophenyl)pyridinyl.
[0088] The term "heteroaryl" as used herein include aromatic ring
systems, including, but not limited to, monocyclic, bicyclic and
tricyclic rings, and have 3 to 12 atoms including at least one
heteroatom, such as nitrogen, oxygen, or sulfur. For purposes of
exemplification, which should not be construed as limiting the
scope of this invention: azaindolyl, benzo(b)thienyl,
benzimidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl,
benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl, furanyl,
imidazolyl, imidazopyridinyl, indolyl, indolinyl, indazolyl,
isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl,
oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl,
pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl,
pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl,
thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl,
thienyl, thiomorpholinyl, triazolyl or tropanyl. The heteroaryl
groups of the invention are substituted with 0, 1, 2, 3, 4 or 5
substituents independently selected from the group consisting of
alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy,
alkoxy, alkenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy,
haloalkoxy, fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio,
fluoroalkylthio, alkenylthio, alkynylthio, sulfonic acid,
alkylsulfonyl, haloalkylsulfonyl, fluoroalkylsulfonyl,
alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl, halo
alkoxysulfonyl, fluoroalkoxysulfonyl, alkenyloxysulfonyl,
alkynyloxysulfonyl, aminosulfonyl, sulfinic acid, alkylsulfinyl,
haloalkylsulfinyl, fluoroalkylsulfinyl, alkenylsulfinyl,
alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl,
fluoroalkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny,
aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl,
fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy,
alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl,
alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, halo
alkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy,
alkynylcarbonyloxy, alkylsulfonyloxy, halo alkylsulfonyloxy,
fluoroalkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy,
haloalkoxysulfonyloxy, fluoroalkoxysulfonyloxy,
alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,
halo alkylsulfinyloxy, fluoroalkylsulfinyloxy, alkenylsulfinyloxy,
alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy,
fluoroalkoxysulfinyloxy, alkenyloxysulfinyloxy,
alkynyloxysulfinyloxy, aminosulfinyloxy, amino, amido,
aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl,
phosphoryl, silyl, silyloxy, and any of said substituents bound to
the heteroaryl group through an alkylene moiety (e.g.
methylene).
[0089] The term "heteroarylene," is art-recognized, and as used
herein pertains to a bidentate moiety obtained by removing two
hydrogen atoms of a heteroaryl ring, as defined above.
[0090] The term "heteroarylalkyl" or "heteroaralkyl" as used herein
means a heteroaryl, as defined herein, appended to the parent
molecular moiety through an alkyl group, as defined herein.
Representative examples of heteroarylalkyl include, but are not
limited to, pyridin-3-ylmethyl and 2-(thien-2-yl)ethyl.
[0091] The term "halo" or "halogen" means --Cl, --Br, --I or
--F.
[0092] The term "haloalkyl" means an alkyl group, as defined
herein, wherein at least one hydrogen is replaced with a halogen,
as defined herein. Representative examples of haloalkyl include,
but are not limited to, chloromethyl, 2-fluoroethyl,
trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.
[0093] The term "fluoroalkyl" means an alkyl group, as defined
herein, wherein all the hydrogens are replaced with fluorines.
[0094] The term "hydroxy" as used herein means an --OH group.
[0095] The term "alkoxy" as used herein means an alkyl group, as
defined herein, appended to the parent molecular moiety through an
oxygen atom. Representative examples of alkoxy include, but are not
limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy,
tert-butoxy, pentyloxy, and hexyloxy. The terms "alkenyloxy",
"alkynyloxy", "carbocyclyloxy", and "heterocyclyloxy" are likewise
defined.
[0096] The term "haloalkoxy" as used herein means an alkoxy group,
as defined herein, wherein at least one hydrogen is replaced with a
halogen, as defined herein. Representative examples of haloalkoxy
include, but are not limited to, chloromethoxy, 2-fluoroethoxy,
trifluoromethoxy, and pentafluoroethoxy. The term "fluoroalkyloxy"
is likewise defined.
[0097] The term "aryloxy" as used herein means an aryl group, as
defined herein, appended to the parent molecular moiety through an
oxygen. The term "heteroaryloxy" as used herein means a heteroaryl
group, as defined herein, appended to the parent molecular moiety
through an oxygen. The terms "heteroaryloxy" is likewise
defined.
[0098] The term "arylalkoxy" or "arylalkyloxy" as used herein means
an arylalkyl group, as defined herein, appended to the parent
molecular moiety through an oxygen. The term "heteroarylalkoxy" is
likewise defined. Representative examples of aryloxy and
heteroarylalkoxy include, but are not limited to,
2-chlorophenylmethoxy, 3-trifluoromethyl-phenylethoxy, and
2,3-dimethylpyridinylmethoxy.
[0099] The term "sulfhydryl" or "thio" as used herein means a --SH
group.
[0100] The term "alkylthio" as used herein means an alkyl group, as
defined herein, appended to the parent molecular moiety through a
sulfur. Representative examples of alkylthio include, but are not
limited, methylthio, ethylthio, tert-butylthio, and hexylthio. The
terms "haloalkylthio", "fluoroalkylthio", "alkenylthio",
"alkynylthio", "carbocyclylthio", and "heterocyclylthio" are
likewise defined.
[0101] The term "arylthio" as used herein means an aryl group, as
defined herein, appended to the parent molecular moiety through an
sulfur. The term "heteroarylthio" is likewise defined.
[0102] The term "arylalkylthio" or "aralkylthio" as used herein
means an arylalkyl group, as defined herein, appended to the parent
molecular moiety through an sulfur. The term "heteroarylalkylthio"
is likewise defined.
[0103] The term "sulfonyl" as used herein refers to
--S(.dbd.O).sub.2-- group.
[0104] The term "sulfonic acid" as used herein refers to
--S(.dbd.O).sub.2OH.
[0105] The term "alkylsulfonyl" as used herein means an alkyl
group, as defined herein, appended to the parent molecular moiety
through a sulfonyl group, as defined herein. Representative
examples of alkylsulfonyl include, but are not limited to,
methylsulfonyl and ethylsulfonyl. The terms "haloalkylsulfonyl",
"fluoroalkylsulfonyl", "alkenylsulfonyl", "alkynylsulfonyl",
"carbocyclylsulfonyl", "heterocyclylsulfonyl", "arylsulfonyl",
"aralkylsulfonyl", "heteroarylsulfonyl" and "heteroaralkylsulfonyl"
are likewise defined.
[0106] The term "alkoxysulfonyl" as used herein means an alkoxy
group, as defined herein, appended to the parent molecular moiety
through a sulfonyl group, as defined herein. Representative
examples of alkoxysulfonyl include, but are not limited to,
methoxysulfonyl, ethoxysulfonyl and propoxysulfonyl. The terms
"haloalkoxysulfonyl", "fluoroalkoxysulfonyl", "alkenyloxysulfonyl",
"alkynyloxysulfonyl", "carbocyclyloxysulfonyl",
"heterocyclyloxysulfonyl", "aryloxysulfonyl", "aralkyloxysulfonyl",
"heteroaryloxysulfonyl" and "heteroaralkyloxysulfonyl" are likewise
defined.
[0107] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0108] The term "aminosulfonyl" as used herein means an amino
group, as defined herein, appended to the parent molecular moiety
through a sulfonyl group.
[0109] The term "sulfinyl" as used herein refers to --S(.dbd.O)--
group. Sulfinyl groups are as defined above for sulfonyl groups.
The term "sulfinic acid" as used herein refers to
--S(.dbd.O)OH.
[0110] The term "oxy" refers to a --O-- group.
[0111] The term "carbonyl" as used herein means a --C(.dbd.O)--
group.
[0112] The term "thiocarbonyl" as used herein means a --C(.dbd.S)--
group.
[0113] The term "formyl" as used herein means a --C(.dbd.O)H
group.
[0114] The term "alkylcarbonyl" as used herein means an alkyl
group, as defined herein, appended to the parent molecular moiety
through a carbonyl group, as defined herein. Representative
examples of alkylcarbonyl include, but are not limited to, acetyl,
1-oxopropyl, 2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl.
The terms "haloalkylcarbonyl", "fluoroalkylcarbonyl",
"alkenylcarbonyl", "alkynylcarbonyl", "carbocyclylcarbonyl",
"heterocyclylcarbonyl", "arylcarbonyl", "aralkylcarbonyl",
"heteroarylcarbonyl", and "heteroaralkylcarbonyl" are likewise
defined.
[0115] The term "carboxy" as used herein means a --CO.sub.2H
group.
[0116] The term "alkoxycarbonyl" as used herein means an alkoxy
group, as defined herein, appended to the parent molecular moiety
through a carbonyl group, as defined herein. Representative
examples of alkoxycarbonyl include, but are not limited to,
methoxycarbonyl, ethoxycarbonyl, and tert-butoxycarbonyl. The terms
"haloalkoxycarbonyl", "fluoroalkoxycarbonyl", "alkenyloxycarbonyl",
"alkynyloxycarbonyl", "carbocyclyloxycarbonyl",
"heterocyclyloxycarbonyl", "aryloxycarbonyl", "aralkyloxycarbonyl",
"heteroaryloxycarbonyl", and "heteroaralkyloxycarbonyl" are
likewise defined.
[0117] The term "alkylcarbonyloxy" as used herein means an
alkylcarbonyl group, as defined herein, appended to the parent
molecular moiety through an oxygen atom. Representative examples of
alkylcarbonyloxy include, but are not limited to, acetyloxy,
ethylcarbonyloxy, and tert-butylcarbonyloxy. The terms
"haloalkylcarbonyloxy", "fluoroalkylcarbonyloxy",
"alkenylcarbonyloxy", "alkynylcarbonyloxy",
"carbocyclylcarbonyloxy", "heterocyclylcarbonyloxy",
"arylcarbonyloxy", "aralkylcarbonyloxy", "heteroarylcarbonyloxy",
and "heteroaralkylcarbonyloxy" are likewise defined.
[0118] The term "alkylsulfonyloxy" as used herein means an
alkylsulfonyl group, as defined herein, appended to the parent
molecular moiety through an oxygen atom. The terms
"haloalkylsulfonyloxy", "fluoroalkylsulfonyloxy",
"alkenylsulfonyloxy", "alkynylsulfonyloxy",
"carbocyclylsulfonyloxy", "heterocyclylsulfonyloxy",
"arylsulfonyloxy", "aralkylsulfonyloxy", "heteroarylsulfonyloxy",
"heteroaralkylsulfonyloxy", "haloalkoxysulfonyloxy",
"fluoroalkoxysulfonyloxy", "alkenyloxysulfonyloxy",
"alkynyloxysulfonyloxy", "carbocyclyloxysulfonyloxy",
"heterocyclyloxysulfonyloxy", "aryloxysulfonyloxy",
"aralkyloxysulfonyloxy", "heteroaryloxysulfonyloxy" and
"heteroaralkyloxysulfonyloxy"
[0119] The term "amino" as used herein refers to --NH.sub.2 and
substituted derivatives thereof wherein one or both of the
hydrogens are independently replaced with substituents selected
from the group consisting of alkyl, haloalkyl, fluoroalkyl,
alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, alkylcarbonyl, haloalkylcarbonyl,
fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl,
carbocyclylcarbonyl, heterocyclylcarbonyl, arylcarbonyl,
aralkylcarbonyl, heteroarylcarnbonyl, heteroaralkylcarbonyl and the
sulfonyl and sulfinyl groups defined above; or when both hydrogens
together are replaced with an alkylene group (to form a ring which
contains the nitrogen). Representative examples include, but are
not limited to methylamino, acetylamino, and dimethylamino.
[0120] The term "amido" as used herein means an amino group, as
defined herein, appended to the parent molecular moiety through a
carbonyl.
[0121] The term "cyano" as used herein means a --C.ident.N
group.
[0122] The term "nitro" as used herein means a --NO.sub.2
group.
[0123] The term "azido" as used herein means a --N.sub.3 group.
[0124] The term "phosphinyl" as used herein includes --PH.sub.3 and
substituted derivatives thereof wherein one, two or three of the
hydrogens are independently replaced with substituents selected
from the group consisting of alkyl, haloalkyl, fluoroalkyl,
alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, alkoxy, haloalkoxy, fluoroalkyloxy,
alkenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, aryloxy,
aralkyloxy, heteroaryloxy, heteroaralkyloxy, and amino.
[0125] The term "phosphoryl" as used herein refers to
--P(.dbd.O)OH.sub.2 and substituted derivatives thereof wherein one
or both of the hydroxyls are independently replaced with
substituents selected from the group consisting of alkyl,
haloalkyl, fluoroalkyl, alkenyl, alkynyl, carbocyclyl,
heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkoxy,
haloalkoxy, fluoroalkyloxy, alkenyloxy, alkynyloxy, carbocyclyloxy,
heterocyclyloxy, aryloxy, aralkyloxy, heteroaryloxy,
heteroaralkyloxy, and amino.
[0126] The term "silyl" as used herein includes H.sub.3Si-- and
substituted derivatives thereof wherein one, two or three of the
hydrogens are independently replaced with substituents selected
from alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl, carbocyclyl,
heterocyclyl, aryl, aralkyl, heteroaryl, and heteroaralkyl.
Representative examples include trimethylsilyl (TMS),
tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TB
S/TBDMS), triisopropylsilyl (TIPS), and
[2-(trimethylsilyl)ethoxy]methyl (SEM).
[0127] The term "silyloxy" as used herein means a silyl group, as
defined herein, is appended to the parent molecule through an
oxygen atom.
[0128] The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations.
[0129] As used herein, the term "administering" means providing a
pharmaceutical agent or composition to a subject, and includes, but
is not limited to, administering by a medical professional and
self-administering.
[0130] As used herein, the phrase "pharmaceutically acceptable"
refers to those agents, compounds, materials, compositions, and/or
dosage forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0131] As used herein, the phrase "pharmaceutically-acceptable
carrier" means a pharmaceutically-acceptable material, composition
or vehicle, such as a liquid or solid filler, diluent, excipient,
or solvent encapsulating material, involved in carrying or
transporting an agent from one organ, or portion of the body, to
another organ, or portion of the body. Each carrier must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,
polycarbonates and/or polyanhydrides; and (22) other non-toxic
compatible substances employed in pharmaceutical formulations.
[0132] As used herein, the phrase "pharmaceutically-acceptable
salts" refers to the relatively non-toxic, inorganic and organic
salts of compounds.
[0133] The term "viral infection" as used herein refers to
infection by a viral pathogen wherein there is clinical evidence of
the infection based on symptoms or based on the demonstration of
the presence of the viral pathogen in a biological sample from the
individual. As used herein an "individual" refers to an animal,
preferably a mammal, including both non-human mammals and humans,
and more preferably, refers to a human.
[0134] "Treatment of a viral infection" as used herein encompasses
alleviating, reducing the frequency of, or eliminating one or more
symptoms of the infection and/or a reducing the viral load.
[0135] As used herein, the term "subject" means a human or
non-human animal selected for treatment or therapy.
[0136] As used herein, the phrase "subject suspected of having"
means a subject exhibiting one or more clinical indicators of a
disease or condition.
[0137] As used herein, the phrase "subject in need thereof" means a
subject identified as in need of a therapy or treatment of the
invention.
[0138] As used herein, the phrase "therapeutic effect" refers to a
local or systemic effect in animals, particularly mammals, and more
particularly humans, caused by an agent. The phrases
"therapeutically-effective amount" and "effective amount" mean the
amount of an agent that produces some therapeutically useful effect
on the symptoms of the viral infection and/or a reduction in viral
load. A therapeutically effective amount includes an amount of an
agent that produces some desired local or systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. For
example, certain agents used in the methods of the invention may be
administered in a sufficient amount to produce a reasonable
benefit/risk ratio applicable to such treatment.
[0139] As used herein, the term "treating" a disease in a subject
or "treating" a subject having or suspected of having a disease
refers to subjecting the subject to a pharmaceutical treatment,
e.g., the administration of an agent, such that at least one
symptom of the disease is decreased or prevented from
worsening.
[0140] As used herein, the phrase "inhibiting replication" means to
reduce replication of a virus in a host cell by about 20%, about
30%, about 40%, about 50%, about 60%, about 70%, about 80%, about
90%, or about 95%, in comparison to an untreated cell. In certain
embodiments, "inhibiting replication" means to reduce replication
of a virus in a host cell by at least about 50%, in comparison to
an untreated cell.
Compounds of the Invention
[0141] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
chemically-protected form, enantiomer or stereoisomer thereof,
wherein the compound is represented by formula I
##STR00005##
wherein, independently for each occurrence,
##STR00006##
is optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted aralkyl, or optionally substituted
heteroaralkyl;
##STR00007##
is optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted aralkyl, or optionally substituted
heteroaralkyl;
[0142] L.sup.1 is a bond, --NR--, --O--, CR.sub.2--, --S--,
--(CR.sub.2).sub.2--, --C(O)--NR--, --NR--C(O)--, --C(O)O--,
--O--C(O)--, --OCR.sub.2--, --CR.sub.2O--, --NR--CR.sub.2--, or
--CR.sub.2--NR--;
[0143] L.sup.2 is a bond, --NR--, --O--, CR.sub.2--, --S--,
--(CR.sub.2).sub.2--, --C(O)--NR--, --NR--C(O)--, --C(O)O--,
--O--C(O)--, --OCR.sub.2--, --CR.sub.2O--, --NR--CR.sub.2--, or
--CR.sub.2--NR--;
[0144] X is H or halo; and
[0145] R is H, alkyl, aryl, or aralkyl.
[0146] In certain embodiments, the invention relates to a compound,
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
chemically-protected form, enantiomer or stereoisomer thereof,
wherein the compound is represented by formula II
##STR00008##
wherein, independently for each occurrence,
##STR00009##
is optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted aralkyl, or optionally substituted
heteroaralkyl;
##STR00010##
is optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted aralkyl, or optionally substituted
heteroaralkyl;
[0147] L.sup.1 is a bond, --NR--, --O--, CR.sub.2--, --S--,
--(CR.sub.2).sub.2--, --C(O)--NR--, --NR--C(O)--, --C(O)O--,
--O--C(O)--, --OCR.sub.2--, --CR.sub.2O--, --NR--CR.sub.2--, or
--CR.sub.2--NR--;
[0148] L.sup.2 is a bond, --NR--, --O--, CR.sub.2--, --S--,
--(CR.sub.2).sub.2--, --C(O)--NR--, --NR--C(O)--, --C(O)O--,
--O--C(O)--, --OCR.sub.2--, --CR.sub.2O--, --NR--CR.sub.2--, or
--CR.sub.2--NR--; and
[0149] R is H, alkyl, aryl, or aralkyl,
[0150] provided the compound is not
##STR00011##
[0151] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00012##
is substituted or unsubstituted aryl. In certain embodiments, the
invention relates to any one of the aforementioned compounds,
wherein
##STR00013##
is substituted or unsubstituted phenyl. In certain embodiments, the
invention relates to any one of the aforementioned compounds,
wherein
##STR00014##
is substituted or unsubstituted naphthyl.
[0152] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00015##
is substituted or unsubstituted aralkyl. In certain embodiments,
the invention relates to any one of the aforementioned compounds,
wherein
##STR00016##
is substituted or unsubstituted benzyl.
[0153] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00017##
is substituted or unsubstituted aryl. In certain embodiments, the
invention relates to any one of the aforementioned compounds,
wherein
##STR00018##
is substituted or unsubstituted phenyl.
[0154] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00019##
is substituted or unsubstituted heteroaryl. In certain embodiments,
the invention relates to any one of the aforementioned compounds,
wherein
##STR00020##
is substituted or unsubstituted pyrazolyl or imidazolyl.
[0155] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00021##
is substituted with one or more substituents selected from the
group consisting of alkoxy, alkyl, --C(O)NR.sub.2, --C(O)OR,
fluoroalkyl, fluoroalkyloxy, aminoalkyl, hydroxyalkyl, halo, cyano,
nitro, aryl, heteroaryl, aralkyl, heteroaralkyl,
--(OCH.sub.2CH.sub.2).sub.n--NH.sub.2 (wherein n is 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10), heterocyclylalkyl, --SO.sub.2NR.sub.2,
aminoalkyl, aryloxy, and heteroaryloxy. In certain embodiments, the
invention relates to any one of the aforementioned compounds,
wherein
##STR00022##
is substituted with one or more substituents selected from the
group consisting of alkoxy, fluoroalkyl, fluoroalkyloxy,
aminoalkyl, hydroxyalkyl, halo, cyano, and nitro. In certain
embodiments, the invention relates to any one of the aforementioned
compounds, wherein
##STR00023##
or
##STR00024##
is substituted with one or more substituents selected from the
group consisting of --OCF.sub.3 and --CF.sub.3. In certain
embodiments, the invention relates to any one of the aforementioned
compounds, wherein
##STR00025##
is substituted with one or more substituents selected from the
group consisting of heteroaralkyl, heterocyclylalkyl,
--SO.sub.2NR.sub.2, and aminoalkyl. In certain embodiments, the
invention relates to any one of the aforementioned compounds,
wherein
##STR00026##
is substituted with one or more substituents selected from the
group consisting of heteroaralkyl, heterocyclylalkyl,
--SO.sub.2NR.sub.2, and aminoalkyl.
[0156] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00027##
is substituted with a fluorescent group. In certain embodiments,
the invention relates to any one of the aforementioned compounds,
wherein
##STR00028##
is substituted with fluorescein isothiocyanate. In certain
embodiments, the invention relates to any one of the aforementioned
compounds, wherein
##STR00029##
is substituted with boron-dipyrromethene.
[0157] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00030##
is substituted with a targeting moiety. In certain embodiments, the
invention relates to any one of the aforementioned compounds,
wherein
##STR00031##
is substituted with biotin.
[0158] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein
##STR00032##
is substituted with a solubilizing group. In certain embodiments,
the invention relates to any one of the aforementioned compounds,
wherein
##STR00033##
is substituted with heterocycloalkyl. In certain embodiments, the
invention relates to any one of the aforementioned compounds,
wherein
##STR00034##
is substituted with alkyl-substituted heterocycloalkyl. In certain
embodiments, the invention relates to any one of the aforementioned
compounds, wherein
##STR00035##
is substituted with heterocycloalkyl-substituted alkyl. In certain
embodiments, the invention relates to any one of the aforementioned
compounds, wherein
##STR00036##
is substituted with alkyl-substituted heterocycloalkyl alkyl. In
certain embodiments, the invention relates to any one of the
aforementioned compounds, wherein
##STR00037##
is substituted with alkoxy. In certain embodiments, the invention
relates to any one of the aforementioned compounds, wherein
##STR00038##
is substituted with heterycycloalkyl-substituted alkoxy. In certain
embodiments, the invention relates to any one of the aforementioned
compounds, wherein
##STR00039##
is substituted with haloalkyl. In certain embodiments, the
invention relates to any one of the aforementioned compounds,
wherein
##STR00040##
is substituted with fluoroalkyl. In certain embodiments, the
invention relates to any one of the aforementioned compounds,
wherein
##STR00041##
is substituted with trifluoromethyl.
[0159] In certain embodiments, the fluorescent group, the targeting
moiety, or the solubilizing moiety is covalently bonded to a
linker, which, in turn, is covalently bonded to
##STR00042##
In certain embodiments, the linker is an oligooxyalkylene chain. In
certain embodiments, the linker is an oligooxyethylene chain. In
certain embodiments, covalent bond linking the linker to the
compound or to the fluorescent group, the targeting moiety, or the
solubilizing group is an amide bond.
[0160] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein L.sup.1 is --NR-- or --O--.
In certain embodiments, the invention relates to any one of the
aforementioned compounds, wherein L.sup.1 is --NR--. In certain
embodiments, the invention relates to any one of the aforementioned
compounds, wherein L.sup.1 is --NH--.
[0161] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein L.sup.2 is --NR-- or --O--.
In certain embodiments, the invention relates to any one of the
aforementioned compounds, wherein L.sup.2 is --NR--. In certain
embodiments, the invention relates to any one of the aforementioned
compounds, wherein L.sup.2 is --NH--.
[0162] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein L.sup.2 is a bond.
[0163] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein the compound is selected from
the group consisting of
##STR00043##
[0164] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein the compound is a compound
defined in FIG. 17, FIG. 18, FIG. 19, FIG. 20, FIG. 26, FIG. 27,
FIG. 28, or FIG. 29.
[0165] In certain embodiments, the invention relates to any one of
the aforementioned compounds, wherein the compound is selected from
the group consisting of
##STR00044##
Therapeutic Methods of the Invention
[0166] In certain embodiments, the invention relates to a method of
inhibiting entry of a virus into a host cell comprising contacting
the host cell with an effective amount of any one of the
aforementioned compounds or
##STR00045##
[0167] In certain embodiments, the invention relates to a method of
inhibiting replication of a virus in a host cell comprising
contacting the host cell with an effective amount of any one of the
aforementioned compounds or
##STR00046##
[0168] In certain embodiments, the invention relates to a method of
inhibiting fusion of a virus to the cell membrane of a host cell
comprising contacting the host cell with an effective amount of any
one of the aforementioned compounds or
##STR00047##
[0169] In certain embodiments, the invention relates to any of the
aforementioned methods, wherein the host cell is contacted with the
compound before exposure to the virus.
[0170] In certain embodiments, the invention relates to any of the
aforementioned methods, wherein the host cell is contacted with the
compound after exposure to the virus.
[0171] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the virus is of family
Flaviviridae.
[0172] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the virus is of genus
Flavivirus, Pestivirus, or Hepacivirus. In certain embodiments, the
invention relates to any one of the aforementioned methods, wherein
the virus is of genus Flavivirus.
[0173] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the virus is dengue virus
(DENV).
[0174] In certain embodiments, the invention relates to a method of
treating or preventing a viral infection in a subject comprising
administering to the subject, (e.g., a subject in need thereof), an
effective amount of any one of the aforementioned compounds.
[0175] In certain embodiments, the invention relates to any of the
aforementioned methods, wherein the compound is administered to the
subject before exposure to a virus; and the virus causes the viral
infection.
[0176] In certain embodiments, the invention relates to any of the
aforementioned methods, wherein the compound is administered to the
subject after exposure to a virus; and the virus causes the viral
infection.
[0177] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the viral infection is a result
of a virus of family Flaviviridae.
[0178] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the viral infection is a result
of a virus of genus Flavivirus, Pestivirus, or Hepacivirus. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the viral infection is a result of
a virus of genus Flavivirus. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the viral
infection is a result of dengue virus (DENV).
[0179] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the viral infection is selected
from the group consisting of: Dengue fever, Japanese encephalitis,
Kyasanur Forest disease, Murray Valley encephalitis, St. Louis
encephalitis, Tick-borne encephalitis, West Nile encephalitis,
Yellow fever, and Hepatitis C.
[0180] The methods of the invention are useful for treating a
subject in need thereof. A subject in need thereof is a subject
having or at risk of having an enveloped virus infection. In its
broadest sense, the terms "treatment" or "to treat" refer to both
therapeutic and prophylactic treatments. If the subject in need of
treatment is experiencing a condition (i.e., has or is having a
particular condition), then "treating the condition" refers to
ameliorating, reducing or eliminating one or more symptoms arising
from the condition. If the subject in need of treatment is one who
is at risk of having a condition, then treating the subject refers
to reducing the risk of the subject having the condition or, in
other words, decreasing the likelihood that the subject will
develop an infectious disease to the virus, as well as to a
treatment after the subject has been infected in order to fight the
infectious disease, e.g., reduce or eliminate it altogether or
prevent it from becoming worse.
[0181] Thus the invention encompasses the use of the compounds
described herein alone or in combination with other therapeutics
for the treatment of a subject having or at risk of having a viral
infection, e.g., an enveloped viral infection. A "subject having an
enveloped viral infection" is a subject that has had contact with a
virus. Thus the virus has invaded the body of the subject. The word
"invade" as used herein refers to contact by the virus with an
external surface of the subject, e.g., skin or mucosal membranes
and/or refers to the penetration of the external surface of the
subject by the virus. A subject at risk of having an enveloped
virus infection is one that has been exposed to or may become
exposed to an enveloped virus or a geographical area in which an
enveloped viral infection has been reported. Further risks include
close contact with a human or non-human primate or their tissues
infected with the virus. Such persons include laboratory or
quarantine facility workers who handle non-human primates that have
been associated with the disease. In addition, hospital staff and
family members who care for patients with the disease are at risk
if they do not use proper barrier nursing techniques.
[0182] As used herein, a subject includes humans and non-human
animals such as non-human primates, dogs, cats, sheep, goats, cows,
pigs, horses and rodents.
[0183] In certain embodiments, the methods of the invention are
useful for treating infection with enveloped viruses. Viruses are
small infectious agents which contain a nucleic acid core and a
protein coat, but are not independently living organisms. A virus
cannot multiply in the absence of a living cell within which it can
replicate. Viruses enter specific living cells either by transfer
across a membrane or direct injection and multiply, causing
disease. The multiplied virus can then be released and infect
additional cells. Some viruses are DNA-containing viruses and
others are RNA-containing viruses. The genomic size, composition
and organization of viruses show tremendous diversity.
[0184] As used herein, an "enveloped" virus is an animal virus
which possesses a membrane or `envelope`, which is a lipid bilayer
containing viral proteins. The envelope proteins of a virus play a
pivotal role in its lifecycle. They participate in the assembly of
the infectious particle and also play a crucial role in virus entry
by binding to a receptor present on the host cell and inducing
fusion between the viral envelope and a membrane of the host cell.
Enveloped viruses can be either spherical or filamentous
(rod-shaped) and include but are not limited to filoviruses, such
as Ebola virus or Marburg virus, Lassa virus, Arboroviruses such as
Togaviruses, flaviviruses (such as hepatitis-C virus),
bunyaviruses, and Arenaviruses, Orthomyxoviridae, Paramyxoviridae,
poxvirus, herpesvirus, hepadnavirus, Rhabdovirus, Bornavirus, and
Arterivirus.
[0185] Flaviviridae is a member of the family of (+)-sense RNA
enveloped viruses. Flaviviridae includes Flavivirus, Pestivirus,
and Hepacivirus. The Flavivirus genus includes yellow fever virus,
dengue fever virus, West Nile virus, and Japanese encaphilitis (JE)
virus. Major diseases caused by viruses in the Flaviviridae family
include: Dengue fever, Japanese encephalitis, Kyasanur Forest
disease, Murray Valley encephalitis, St. Louis encephalitis,
Tick-borne encephalitis, West Nile encephalitis, Yellow fever, and
Hepatitis C. The Pestivirus genus includes the three serotypes of
bovine viral diarrhea, but no known human pathogens. Genus
Hepacivirus consists of hepatitis C virus and hepatitis C-like
viruses.
[0186] A yellow fever virus infection is characterized by an
incubation period of 3 to 6 days, during which 5% to 50% of
infected people develop disease. Yellow fever begins with a
nonspecific 1- to 3-day febrile illness, followed by a brief
remission, and then by a life-threatening toxic syndrome
accompanied by epistaxis, other hemorrhagic phenomena, jaundice,
and disseminated intravascular coagulation. Mortality rates for
yellow fever are approximately 20%.
[0187] There are four serotypes of dengue fever virus, all
transmitted by mosquitoes. Dengue fever virus infection may be
asymptomatic or may result in dengue fever. This is generally a
self-limiting febrile illness which occurs after a 4-8 day
incubation period. It has symptoms such as fever, aches and
arthralgia (pain in the joints) which can progress to arthritis
(inflammation of the joints), myositis (inflammation of muscle
tissue) and a discrete macular or maculopapular rash. In this
situation clinical differentiation from other viral illnesses may
not be possible, recovery is rapid, and need for supportive
treatment is minimal. Dengue haemorrhagic fever (DHF) is a
potentially deadly complication. Dengue hemorrhagic fever commences
with high fever and many of the symptoms of dengue fever, but with
extreme lethargy and drowsiness. The patient has increased vascular
permeability and abnormal homeostasis that can lead to hypovolemia
and hypotension, and in severe cases, result in hypovolemic shock
often complicated by severe internal bleeding.
[0188] The Japanese encephalitis antigenic complex includes Alfuy,
Japanese encephalitis, Kokobera, Koutango, Kunjin, Murray Valley
encephalitis, St. Louis encephalitis, Stratford, Usutu, and West
Nile viruses. These viruses are transmissible by mosquitoes and
many of them can cause febrile, sometimes fatal, illnesses in
humans. West Nile virus is the most widespread of the flaviviruses,
with geographic distribution including Africa and Eurasia. West
Nile virus RNA has been detected in overwintering mosquitoes in New
York City & the geographic range of the virus is increasing in
the USA.
[0189] The genus Pestivirus has been divided into bovine viral
diarrhea virus (BVDV), classical swine fever virus (CSFV), and
border disease virus (BDV). Infection with BVDV results in a
variety of diseases ranging from subclinical to highly fatal. Many
BVDV viruses cause only clinically mild disease in nonpregnant
adult cattle. Prenatal infection can cause congenital malformations
and/or fetal death.
[0190] The Hepacivirus genus includes the hepatitis C virus (HCV).
The majority of cases of HCV infection give rise to an acute
illness, where up to 85% of infections may develop into chronic
hepatitis. Almost all patients develop a vigorous antibody and
cell-mediated immune response which fails to clear the infection
but may contribute towards liver damage.
[0191] In some embodiments, the desired dose of the active agent
will depend on absorption, inactivation, and excretion rates of the
drug as well as the delivery rate of the compound. It is to be
noted that dosage values may also vary with the severity of the
condition to be alleviated. It is to be further understood that for
any particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the person administering or supervising the
administration of the compositions. Typically, dosing will be
determined using techniques known to one skilled in the art.
[0192] The dosage of the active agent may be determined by
reference to the plasma concentrations of the agent. For example,
the maximum plasma concentration (Cmax) and the area under the
plasma concentration-time curve from time 0 to infinity (AUC (0-4))
may be used. Dosages for the invention include those that produce
the above values for Cmax and AUC (0-4) and other dosages resulting
in larger or smaller values for those parameters.
[0193] Actual dosage levels of the active agents may be varied so
as to obtain an amount of the active ingredient which is effective
to achieve the desired therapeutic response for a particular
patient, composition, and mode of administration, without being
toxic to the patient.
[0194] The selected dosage level will depend upon a variety of
factors including the activity of the particular agent employed,
the route of administration, the time of administration, the rate
of excretion or metabolism of the particular compound being
employed, the duration of the treatment, other drugs, compounds
and/or materials used in combination with the particular compound
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0195] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could prescribe and/or administer doses of the agents
of the invention employed in the pharmaceutical composition at
levels lower than that required in order to achieve the desired
therapeutic effect and gradually increase the dosage until the
desired effect is achieved.
[0196] In general, a suitable daily dose of an agent of the
invention will be that amount of the agent (e.g., the compound)
which is the lowest dose effective to produce a therapeutic effect.
Such an effective dose will generally depend upon the factors
described above.
[0197] If desired, the effective daily dose of the agent may be
administered as two, three, four, five, six or more sub-doses
administered separately at appropriate intervals throughout the
day, optionally, in unit dosage forms.
[0198] The precise time of administration and amount of any
particular agent that will yield the most effective treatment in a
given patient will depend upon the activity, pharmacokinetics, and
bioavailability of a particular agent, physiological condition of
the patient (including age, sex, disease type and stage, general
physical condition, responsiveness to a given dosage and type of
medication), route of administration, and the like. The guidelines
presented herein may be used to optimize the treatment, e.g.,
determining the optimum time and/or amount of administration, which
will require no more than routine experimentation consisting of
monitoring the subject and adjusting the dosage and/or timing.
[0199] While the subject is being treated, the health of the
subject may be monitored by measuring one or more of the relevant
indices at predetermined times during a 24-hour period. All aspects
of the treatment, including supplements, amounts, times of
administration and formulation, may be optimized according to the
results of such monitoring. The patient may be periodically
reevaluated to determine the extent of improvement by measuring the
same parameters, the first such reevaluation typically occurring at
the end of four weeks from the onset of therapy, and subsequent
reevaluations occurring every four to eight weeks during therapy
and then every three months thereafter. Therapy may continue for
several months or even years, with a minimum of one month being a
typical length of therapy for humans. Adjustments, for example, to
the amount(s) of agent administered and to the time of
administration may be made based on these reevaluations.
[0200] Treatment may be initiated with smaller dosages which are
less than the optimum dose of the compound. Thereafter, the dosage
may be increased by small increments until the optimum therapeutic
effect is attained. In addition, the combined use of an antiviral
agent of the invention and a second agent, e.g. another agent
useful for the treatment or prevention of viral infections, may
reduce the required dosage for any individual agent because the
onset and duration of effect of the different compounds and/or
agents may be complimentary.
[0201] Many of the compounds used in the methods of the invention
may be provided as salts with pharmaceutically compatible
counterions (i.e., pharmaceutically acceptable salts). A
"pharmaceutically acceptable salt" means any non-toxic salt that,
upon administration to a recipient, is capable of providing, either
directly or indirectly, a compound or a prodrug of a compound of
this invention. A "pharmaceutically acceptable counterion" is an
ionic portion of a salt that is not toxic when released from the
salt upon administration to a recipient. Pharmaceutically
compatible salts may be formed with many acids, including but not
limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic,
succinic, etc. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms.
[0202] Acids commonly employed to form pharmaceutically acceptable
salts include inorganic acids such as hydrogen bisulfide,
hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric
acid, as well as organic acids such as para-toluenesulfonic,
salicylic, tartaric, bitartaric, ascorbic, maleic, besylic,
fumaric, gluconic, glucuronic, formic, glutamic, methanesulfonic,
ethanesulfonic, benzenesulfonic, lactic, oxalic,
para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and
acetic acid, and related inorganic and organic acids. Such
pharmaceutically acceptable salts thus include sulfate,
pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,
monohydrogenphosphate, dihydrogenphosphate, metaphosphate,
pyrophosphate, chloride, bromide, iodide, acetate, propionate,
decanoate, caprylate, acrylate, formate, isobutyrate, caprate,
heptanoate, propiolate, oxalate, malonate, succinate, suberate,
sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,
benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,
hydroxybenzoate, methoxybenzoate, phthalate, terephthalate,
sulfonate, xylenesulfonate, phenylacetate, phenylpropionate,
phenylbutyrate, citrate, lactate, .beta.-hydroxybutyrate,
glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,
naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the
like salts. Pharmaceutically acceptable acid addition salts include
those formed with mineral acids such as hydrochloric acid and
hydrobromic acid, and those formed with organic acids such as
maleic acid.
[0203] Suitable bases for forming pharmaceutically acceptable salts
with acidic functional groups include, but are not limited to,
hydroxides of alkali metals such as sodium, potassium, and lithium;
hydroxides of alkaline earth metal such as calcium and magnesium;
hydroxides of other metals, such as aluminum and zinc; ammonia, and
organic amines, such as unsubstituted or hydroxy-substituted mono-,
di-, or trialkylamines; dicyclohexylamine; tributyl amine;
pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine;
mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), such as mono-,
bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or
tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy
lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine,
or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids
such as arginine, lysine, and the like.
[0204] Certain compounds used in methods of the invention and their
salts may exist in more than one crystal form and the invention
includes each crystal form and mixtures thereof.
[0205] Certain compounds used in methods of the invention and their
salts may also exist in the form of solvates, for example hydrates,
and the invention includes each solvate and mixtures thereof.
[0206] Certain compounds used in methods of the invention may
contain one or more chiral centers, and exist in different
optically active forms. When compounds of the invention contain one
chiral center, the compounds exist in two enantiomeric forms and
the invention includes both enantiomers and mixtures of
enantiomers, such as racemic mixtures. The enantiomers may be
resolved by methods known to those skilled in the art, for example
by formation of diastereoisomeric salts which may be separated, for
example, by crystallization; formation of diastereoisomeric
derivatives or complexes which may be separated, for example, by
crystallization, gas-liquid or liquid chromatography; selective
reaction of one enantiomer with an enantiomer-specific reagent, for
example enzymatic esterification; or gas-liquid or liquid
chromatography in a chiral environment, for example on a chiral
support for example silica with a bound chiral ligand or in the
presence of a chiral solvent. It will be appreciated that where the
desired enantiomer is converted into another chemical entity by one
of the separation procedures described above, a further step may be
used to liberate the desired enantiomeric form. Alternatively,
specific enantiomers may be synthesized by asymmetric synthesis
using optically active reagents, substrates, catalysts or solvents,
or by converting one enantiomer into the other by asymmetric
transformation.
[0207] When a compound used in the methods of the invention
contains more than one chiral center, it may exist in
diastereoisomeric forms. The diastereoisomeric compounds may be
separated by methods known to those skilled in the art, for example
chromatography or crystallization and the individual enantiomers
may be separated as described above. The invention includes each
diastereoisomer of compounds of the invention and mixtures
thereof.
[0208] Certain compounds used in methods of the invention may exist
in different tautomeric forms or as different geometric isomers,
and the invention includes each tautomer and/or geometric isomer of
compounds of the invention and mixtures thereof.
[0209] Certain compounds used in methods of the invention may exist
in different stable conformational forms which may be separable.
Torsional asymmetry due to restricted rotation about an asymmetric
single bond, for example because of steric hindrance or ring
strain, may permit separation of different conformers. The
invention includes each conformational isomer of compounds of the
invention and mixtures thereof.
[0210] Certain compounds used in methods of the invention may exist
in zwitterionic form and the invention includes each zwitterionic
form of compounds of the invention and mixtures thereof.
[0211] The invention also includes methods of using pro-drugs. As
used herein the term "pro-drug" refers to an agent which is
converted into the parent drug in vivo by some physiological
chemical process (e.g., a prodrug on being brought to the
physiological pH is converted to the desired drug form). Pro-drugs
are often useful because, in some situations, they may be easier to
administer than the parent drug. They may, for instance, be
bioavailable by oral administration whereas the parent drug is not.
The prodrug may also have improved solubility in pharmacological
compositions over the parent drug. An example, without limitation,
of a pro-drug would be a compound of the invention wherein it is
administered as an ester (the "pro-drug") to facilitate transmittal
across a cell membrane where water solubility is not beneficial,
but then it is metabolically hydrolyzed to the carboxylic acid once
inside the cell where water solubility is beneficial. Pro-drugs
have many useful properties. For example, a pro-drug may be more
water soluble than the ultimate drug, thereby facilitating
intravenous administration of the drug. A pro-drug may also have a
higher level of oral bioavailability than the ultimate drug. After
administration, the prodrug is enzymatically or chemically cleaved
to deliver the ultimate drug in the blood or tissue.
[0212] Exemplary pro-drugs upon cleavage release the corresponding
free acid, and such hydrolyzable ester-forming residues of the
compounds of this invention include but are not limited to
carboxylic acid substituents (e.g., --C(O).sub.2H or a moiety that
contains a carboxylic acid) wherein the free hydrogen is replaced
by (C.sub.1-C.sub.4)alkyl, (C.sub.2-C.sub.12)alkanoyloxymethyl,
(C.sub.4-C.sub.9)1-(alkanoyloxy)ethyl,
1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms,
alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms,
1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,
1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon
atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon
atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon
atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,
di-N,N--(C.sub.1-C.sub.2)alkylamino(C.sub.2-C.sub.3)alkyl (such as
.beta.-dimethylaminoethyl), carbamoyl-(C.sub.1-C.sub.2)alkyl,
N,N-di(C.sub.1-C.sub.2)-alkylcarbamoyl-(C.sub.1-C.sub.2)alkyl and
piperidino-, pyrrolidino- or morpholino(C.sub.2-C.sub.3)alkyl.
[0213] Other exemplary pro-drugs release an alcohol or amine of a
compound of the invention wherein the free hydrogen of a hydroxyl
or amine substituent is replaced by
(C.sub.1-C.sub.6)alkanoyloxymethyl,
1-((C.sub.1-C.sub.6)alkanoyloxy)ethyl,
1-methyl-1-((C.sub.1-C.sub.6)alkanoyloxy)ethyl,
(C.sub.1-C.sub.6)alkoxycarbonyl-oxymethyl,
N--(C.sub.1-C.sub.6)alkoxycarbonylamino-methyl, succinoyl,
(C.sub.1-C.sub.6)alkanoyl, .alpha.-amino(C.sub.1-C.sub.4)alkanoyl,
arylactyl and .alpha.-aminoacyl, or
.alpha.-aminoacyl-.alpha.-aminoacyl wherein said .alpha.-aminoacyl
moieties are independently any of the naturally occurring L-amino
acids found in proteins, --P(O)(OH).sub.2,
--P(O)(O(C.sub.1-C.sub.6)alkyl).sub.2 or glycosyl (the radical
resulting from detachment of the hydroxyl of the hemiacetal of a
carbohydrate).
[0214] The phrase "protecting group" as used herein means temporary
substituents which protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 2.sup.nd ed.; Wiley: New York, 1991). Protected
forms of the inventive compounds are included within the scope of
this invention.
[0215] The term "chemically protected form," as used herein,
pertains to a compound in which one or more reactive functional
groups are protected from undesirable chemical reactions, that is,
are in the form of a protected or protecting group (also known as a
masked or masking group). It may be convenient or desirable to
prepare, purify, and/or handle the active compound in a chemically
protected form.
[0216] By protecting a reactive functional group, reactions
involving other unprotected reactive functional groups can be
performed, without affecting the protected group; the protecting
group may be removed, usually in a subsequent step, without
substantially affecting the remainder of the molecule. See, for
example, Protective Groups in Organic Synthesis (T. Green and P.
Wuts, Wiley, 1991), and Protective Groups in Organic Synthesis (T.
Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999).
[0217] For example, a hydroxy group may be protected as an ether
(--OR) or an ester (--OC(.dbd.O)R), for example, as: a t-butyl
ether; a benzyl, benzhydryl(diphenylmethyl), or
trityl(triphenylmethyl) ether; a trimethylsilyl or
t-butyldimethylsilyl ether; or an acetyl ester
(--OC(.dbd.O)CH.sub.3, --OAc).
[0218] For example, an aldehyde or ketone group may be protected as
an acetal or ketal, respectively, in which the carbonyl group
(C(.dbd.O)) is converted to a diether (C(OR).sub.2), by reaction
with, for example, a primary alcohol. The aldehyde or ketone group
is readily regenerated by hydrolysis using a large excess of water
in the presence of acid.
[0219] For example, an amine group may be protected, for example,
as an amide (--NRC(.dbd.O)R) or a urethane (--NRC(.dbd.O)OR), for
example, as: a methyl amide (--NHC(.dbd.O)CH.sub.3); a benzyloxy
amide (--NHC(.dbd.O)OCH.sub.2C.sub.6H.sub.5NHCbz); as a t-butoxy
amide (--NHC(.dbd.O)OC(CH.sub.3).sub.3, --NHBoc); a
2-biphenyl-2-propoxy amide
(--NHC(.dbd.O)OC(CH.sub.3).sub.2C.sub.6H.sub.4C.sub.6H.sub.5NHBoc),
as a 9-fluorenylmethoxy amide (--NHFmoc), as a 6-nitroveratryloxy
amide (--NHNvoc), as a 2-trimethylsilylethyloxy amide (--NHTeoc),
as a 2,2,2-trichloroethyloxy amide (--NHTroc), as an allyloxy amide
(--NHAlloc), as a 2-(phenylsulfonyl)ethyloxy amide (--NHPsec); or,
in suitable cases (e.g., cyclic amines), as a nitroxide
radical.
[0220] For example, a carboxylic acid group may be protected as an
ester or an amide, for example, as: a benzyl ester; a t-butyl
ester; a methyl ester; or a methyl amide.
[0221] For example, a thiol group may be protected as a thioether
(--SR), for example, as: a benzyl thioether; or an acetamidomethyl
ether (--SCH.sub.2NHC(.dbd.O)CH.sub.3).
Combination Therapy
[0222] In certain embodiments, the invention relates to a method of
co-administering a compound of Formula I and at least one other
therapeutic agent. The compound and other therapeutic agent may be
administered simultaneously or sequentially. When the other
therapeutic agents are administered simultaneously they can be
administered in the same or separate formulations, but are
administered at the same time. The other therapeutic agents are
administered sequentially with one another and with the compounds,
when the administration of the other therapeutic agents and the
compounds is temporally separated. The separation in time between
the administration of these compounds may be a matter of minutes or
it may be longer. Other therapeutic agents include but are not
limited to anti-viral vaccines and anti-viral agents. In some
instances the inhibitors are administered with multiple therapeutic
agents, i.e., 2, 3, 4 or even more different anti-viral agents.
[0223] An anti-viral vaccine is a formulation composed of one or
more viral antigens and one or more adjuvants. The viral antigens
include proteins or fragments thereof as well as whole killed
virus. Adjuvants are well known to those of skill in the art.
[0224] Antiviral agents are compounds that prevent infection of
cells by viruses or replication of the virus within the cell. There
are many fewer antiviral drugs than antibacterial drugs because
viruses are more dependent on host cell factors than bacteria.
There are several stages within the process of viral infection
which can be blocked or inhibited by antiviral agents. These stages
include, attachment of the virus to the host cell (immunoglobulin
or binding peptides), membrane penetration inhibitors, e.g. T-20,
uncoating of the virus (e.g. amantadine), synthesis or translation
of viral mRNA (e.g. interferon), replication of viral RNA or DNA
(e.g. nucleotide analogues), maturation of new virus proteins (e.g.
protease inhibitors), and budding and release of the virus.
[0225] Nucleotide analogues are synthetic compounds that are
similar to nucleotides, but which have an incomplete or abnormal
deoxyribose or ribose group. Once the nucleotide analogues are in
the cell, they are phosphorylated, producing the triphosphate
formed which competes with normal nucleotides for incorporation
into the viral DNA or RNA. Once the triphosphate form of the
nucleotide analogue is incorporated into the growing nucleic acid
chain, it causes irreversible association with the viral polymerase
and thus chain termination. Nucleotide analogues include, but are
not limited to, acyclovir (used for the treatment of herpes simplex
virus and varicella-zoster virus), gancyclovir (useful for the
treatment of cytomegalovirus), idoxuridine, ribavirin (useful for
the treatment of respiratory syncitial virus), dideoxyinosine,
dideoxycytidine, zidovudine (azidothymidine), imiquimod, and
resimiquimod.
[0226] The interferons are cytokines that are secreted by
virus-infected cells as well as immune cells. The interferons
function by binding to specific receptors on cells adjacent to the
infected cells, causing the change in the cell which protects it
from infection by the virus. .alpha.- and .beta.-interferon also
induce the expression of Class I and Class II MHC molecules on the
surface of infected cells, resulting in increased antigen
presentation for host immune cell recognition. .alpha.- and
.beta.-interferons are available as recombinant forms and have been
used for the treatment of chronic hepatitis B and C infection. At
the dosages which are effective for anti-viral therapy, interferons
have severe side effects such as fever, malaise and weight
loss.
[0227] Anti-viral agents that may be useful in the methods of the
invention include but are not limited to immunoglobulins,
amantadine, interferons, nucleotide analogues, and other protease
inhibitors (other than the papain-like cysteine protease
inhibitors--although combinations of papain-like cysteine protease
inhibitors are also useful). Specific examples of anti-viral agents
include but are not limited to Acemannan; Acyclovir; Acyclovir
Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine
Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate; Avridine;
Cidofovir; Cipamfylline; Cytarabine Hydrochloride; Delavirdine
Mesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine;
Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride;
Fiacitabine; Fialuridine; Fosarilate; Foscarnet Sodium; Fosfonet
Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal;
Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone;
Nevirapine; Penciclovir; Pirodavir; Ribavirin; Rimantadine
Hydrochloride; Saquinavir Mesylate; Somantadine Hydrochloride;
Sorivudine; Statolon; Stavudine; Tilorone Hydrochloride;
Trifluridine; Valacyclovir Hydrochloride; Vidarabine; Vidarabine
Phosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine;
Zidovudine; and Zinviroxime.
[0228] Immunoglobulin therapy is used for the prevention of viral
infection. Immunoglobulin therapy for viral infections is different
than bacterial infections, because rather than being
antigen-specific, the immunoglobulin therapy functions by binding
to extracellular virions and preventing them from attaching to and
entering cells which are susceptible to the viral infection. The
therapy is useful for the prevention of viral infection for the
period of time that the antibodies are present in the host. In
general there are two types of immunoglobulin therapies, normal
immunoglobulin therapy and hyper-immunoglobulin therapy. Normal
immune globulin therapy utilizes a antibody product which is
prepared from the serum of normal blood donors and pooled. This
pooled product contains low titers of antibody to a wide range of
human viruses, such as hepatitis A, parvovirus, enterovirus
(especially in neonates). Hyper-immune globulin therapy utilizes
antibodies which are prepared from the serum of individuals who
have high titers of an antibody to a particular virus. Those
antibodies are then used against a specific virus. Another type of
immunoglobulin therapy is active immunization. This involves the
administration of antibodies or antibody fragments to viral surface
proteins.
Pharmaceutical Compositions
[0229] The invention provides pharmaceutical compositions for use
in treating or preventing viral infections in vitro or in vivo. In
one aspect, the invention provides pharmaceutically acceptable
compositions which comprise a therapeutically-effective amount of
one or more of the compounds described above, formulated together
with one or more pharmaceutically acceptable carriers (additives)
and/or diluents. In another aspect, the agents of the invention can
be administered as such, or administered in mixtures with
pharmaceutically acceptable carriers and can also be administered
in conjunction with other agents. Conjunctive therapy thus includes
sequential, simultaneous and separate, or co-administration of one
or more compound of the invention, wherein the therapeutic effects
of the first administered has not entirely disappeared when the
subsequent compound is administered.
[0230] As described in detail below, the pharmaceutical
compositions of the invention may be specially formulated for
administration in solid or liquid form, including those adapted for
the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets, e.g.,
those targeted for buccal, sublingual, and systemic absorption,
boluses, powders, granules, pastes for application to the tongue;
(2) parenteral administration, for example, by subcutaneous,
intramuscular, intravenous or epidural injection as, for example, a
sterile solution or suspension, or sustained-release formulation;
(3) topical application, for example, as a cream, ointment, or a
controlled-release patch or spray applied to the skin; (4)
intravaginally or intrarectally, for example, as a pessary, cream
or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)
nasally.
[0231] As set out above, in certain embodiments, agents of the
invention may be compounds containing a basic functional group,
such as amino or alkylamino, and are, thus, capable of forming
pharmaceutically-acceptable salts with pharmaceutically-acceptable
acids. These salts can be prepared in situ in the administration
vehicle or the dosage form manufacturing process, or through a
separate reaction of a purified compound of the invention in its
free base form with a suitable organic or inorganic acid, and
isolating the salt thus formed during subsequent purification.
Representative salts include the hydrobromide, hydrochloride,
sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,
palmitate, stearate, laurate, benzoate, lactate, phosphate,
tosylate, citrate, maleate, fumarate, succinate, tartrate,
naphthylate, mesylate, glucoheptonate, lactobionate, and
laurylsulphonate salts and the like (see, for example, Berge et al.
(1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
[0232] The pharmaceutically acceptable salts of the subject
compounds include the conventional nontoxic salts or quaternary
ammonium salts of the compounds, e.g., from non-toxic organic or
inorganic acids. For example, such conventional nontoxic salts
include those derived from inorganic acids such as hydrochloride,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like;
and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isothionic, and the like.
[0233] In other cases, the compounds of the invention may be
compounds containing one or more acidic functional groups and,
thus, are capable of forming pharmaceutically-acceptable salts with
pharmaceutically-acceptable bases. These salts can likewise be
prepared in situ in the administration vehicle or the dosage form
manufacturing process, or by separately reacting the purified
compound in its free acid form with a suitable base, such as the
hydroxide, carbonate or bicarbonate of a
pharmaceutically-acceptable metal cation, with ammonia, or with a
pharmaceutically-acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like (see, for example, Berge et al., supra).
[0234] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0235] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0236] The formulations of the compounds of the invention may be
presented in unit dosage form and may be prepared by any methods
well known in the art of pharmacy. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will vary depending upon the host being treated and the
particular mode of administration. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will generally be that amount of the agent which
produces a therapeutic effect.
[0237] In certain embodiments, a formulation of the invention
comprises an excipient, including, but not limited to,
cyclodextrins, liposomes, micelle forming agents, e.g., bile acids,
and polymeric carriers, e.g., polyesters and polyanhydrides; and an
agent of the invention. In certain embodiments, an aforementioned
formulation renders orally bioavailable an agent of the
invention.
[0238] Methods of preparing these formulations or compositions may
include the step of bringing into association a compound of the
invention with the carrier and, optionally, one or more accessory
ingredients.
[0239] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0240] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0241] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0242] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
invention as an active ingredient. A compound of the invention may
also be administered as a bolus, electuary or paste.
[0243] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically-acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, cetyl alcohol, glycerol
monostearate, and non-ionic surfactants; (8) absorbents, such as
kaolin and bentonite clay; (9) lubricants, such a talc, calcium
stearate, magnesium stearate, solid polyethylene glycols, sodium
lauryl sulfate, and mixtures thereof; and (10) coloring agents. In
the case of capsules, tablets and pills, the pharmaceutical
compositions may also comprise buffering agents. Solid compositions
of a similar type may also be employed as fillers in soft and
hard-shelled gelatin capsules using such excipients as lactose or
milk sugars, as well as high molecular weight polyethylene glycols
and the like.
[0244] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0245] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the invention, such as dragees,
capsules, pills and granules, may optionally be scored or prepared
with coatings and shells, such as enteric coatings and other
coatings well known in the pharmaceutical-formulating art. They may
also be formulated so as to provide slow or controlled release of
the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. Compositions of the invention may also be formulated
for rapid release, e.g., freeze-dried. They may be sterilized by,
for example, filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved in sterile water, or some other
sterile injectable medium immediately before use. These
compositions may also optionally contain opacifying agents and may
be of a composition that they release the active ingredient(s)
only, or preferentially, in a certain portion of the
gastrointestinal tract, optionally, in a delayed manner. Examples
of embedding compositions which can be used include polymeric
substances and waxes. The active ingredient can also be in
micro-encapsulated form, if appropriate, with one or more of the
above-described excipients.
[0246] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound.
[0247] Formulations of the invention which are suitable for vaginal
administration also include pessaries, tampons, creams, gels,
pastes, foams or spray formulations containing such carriers as are
known in the art to be appropriate.
[0248] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0249] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0250] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0251] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the invention to the body.
Such dosage forms can be made by dissolving or dispersing the
compound in the proper medium. Absorption enhancers can also be
used to increase the flux of the compound across the skin. The rate
of such flux can be controlled by either providing a rate
controlling membrane or dispersing the compound in a polymer matrix
or gel.
[0252] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0253] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
sugars, alcohols, antioxidants, buffers, bacteriostats, solutes
which render the formulation isotonic with the blood of the
intended recipient or suspending or thickening agents.
[0254] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0255] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0256] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0257] Exemplary formulations comprising agents of the invention
are determined based on various properties including, but not
limited to, chemical stability at body temperature, functional
efficiency time of release, toxicity and optimal dose.
[0258] The preparations of the invention may be given orally,
parenterally, topically, or rectally. They are of course given in
forms suitable for each administration route. For example, they are
administered in tablets or capsule form, by injection, inhalation,
eye lotion, ointment, suppository, administration by injection,
infusion or inhalation; topical by lotion or ointment; and rectal
by suppositories.
[0259] Regardless of the route of administration selected, the
compounds of the invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
invention, are formulated into pharmaceutically-acceptable dosage
forms by conventional methods known to those of skill in the
art.
EXAMPLES
[0260] The invention now being generally described, it will be more
readily understood by reference to the following, which is included
merely for purposes of illustration of certain aspects and
embodiments of the invention, and is not intended to limit the
invention.
Example 1
DENV Replication in Cell Lines Lacking Abl Kinase Activity
[0261] RNAi was used to deplete cells of c-Abl kinase. Knockout
cell lines were also investigated. Cells depleted of c-Abl kinase,
or mouse embryonic fibroblast (MEF) cells deficient in Abl kinase,
showed a significant reduction in viral yield.
[0262] See FIGS. 2 and 3.
Example 2
Reduction of Viral Titer by Kinase Inhibitors
[0263] GNF2 reduced titers by over 1 log unit, while imatinib did
not. See FIG. 4 and FIG. 5. The IC.sub.50 (kinase) for imatinib was
190.+-.6 nM and for GNF2 was 138.+-.5 nM. The EC.sub.90 (dengue
virus serotype 2, DENV2) for imatinib was 10 .mu.M and about 1
.mu.M for GNF2. GNF2 is monoselective for Abl family kinases,
indicating that GNF2 has an additional antiviral target.
Example 3
Order of Addition Experiments
[0264] In order to investigate the role of kinases on DENV entry,
the order of addition of the cells, virus, and inhibitor was
investigated. An inhibitor was either (i) contacted with a virus
for 1 h at 37.degree. C.; (ii) contacted with cells for 1 h at
37.degree. C.; or (iii) contacted with a virus and cells for 1 h at
37.degree. C. The cells were washed to remove virus and inhibitor.
The washed cells were exposed to either DMSO or an inhibitor. Viral
titer was measured at intervals post-infection by harvesting
supernatants and measuring virus production by a plaque formation
assay (PFA). The data indicate that GNF-2 is active prior to viral
entry while imatinib and GNF2 share a post-entry antiviral
activity. See FIG. 6.
Example 4
Investigation of Post-Entry Role of Abl Kinases
[0265] Imatinib does not lower DV titer when pre-incubated with the
virus as compared to a DMSO control. However, treatment of the
virus with either a quinazoline entry inhibitor (see Wang, Q.-Y.,
et al. Antimicrob. Agents Chemother. 2009, 53(5), 1823-1831) or
GNF-2 significantly lowers titer by .about.1 log. Post-treatment of
cells with either Abl kinase inhibitor significantly reduces titer
as well. Data not shown.
[0266] In short, GNF-2 acts at two separate and distinct steps of
DV life cycle to inhibit DENV.
Example 5
SAR
[0267] See FIG. 7, FIG. 8, and FIG. 9.
[0268] About 90 GNF2 analogs were synthesized and screened.
Twenty-three small molecules were identified that inhibit DENV and
have no measurable activity against Abl kinases. The most potent
inhibitors of DENV entry had EC.sub.90 values of 1-15 .mu.M against
all four dengue serotypes.
Example 6
Biotinylation of Inhibitor
[0269] Biotinylated GNF2 was incubated with virus for 45 min at
37.degree. C., then the competing compound was added for an
additional 45 min. See FIG. 10. No pulldown of vesicular stomatitis
virus (VSV) was observed. Importantly, biotinylated GNF2 retains
anti-DENV activity in cell-based assays.
Example 7
Use of Fluorescent Microscopy to Track GNF2-CY5 During DENV
Entry
[0270] Fluorescent microscopy was used to track uptake of
inhibitors during viral entry. See FIG. 13. Importantly, GNF2-CY5
retains its antiviral activity against DENV serotype 2 as compared
to GNF2.
[0271] These studies showed that GNF2-CY5 was not taken up in the
absence of DENV2 (data not shown). Also, GNF2-CY5 co-localized with
the DENV2 E protein (data not shown). Finally, since GNF2 reduced
DENV titers in cell-based assays, the mechanism of inhibition is
not that the compounds block attachment or uptake of the virus.
Example 8
Effect of Kinase Inhibitors on DENV Fusion
[0272] A capsid protection assay was used to measure fusion pore
formation and content mixing of DENV2 with liposomes. See FIG. 14.
Our data indicate that DENV2 cannot complete fusion in the presence
of the inventive inhibitors. See FIG. 15.
[0273] In addition, the data indicate that an inventive compound
does not block association of DENV with liposomes. See FIG. 16.
Example 9
General Methods
[0274] Compounds were synthesized and purified by established
methods to generate multiple series of regioisomeric 4,6- and
2,4-disubstituted pyrimidines. To make focused changes to the
4,6-disubstituted core structure of GNF-2 while holding either the
4-trifluoromethoxy aniline or the aryl benzamido portions constant,
4,6-dichloropyrimidine was first mono-derivatized either under
basic amination conditions or under Suzuki reaction conditions to
prepare mono-substituted pyrimidine intermediates. The two
resulting intermediates were subsequently reacted with amines,
phenols or anilines in a nucleophilic substitution reaction under
various optimized conditions to afford final compounds in good
yields. Regioisomeric 2,4-disubstituted pyrimidines were prepared
in parallel via an analogous synthetic route by holding the C4
position of the pyrimidine constant with a 4-trifluoromethoxy
aniline or a 3-aryl benzamide and varying the C2 substituents. The
compounds were then screened for anti-DENV activity when present at
75 .mu.M during pre-treatment of the inoculum and the 1 hour
infection period. Compounds that lowered DENV2 titer by at least
one log at this concentration were then tested at 25 .mu.M in order
to determine if these compounds were more effective than c-GNF-2,
our initial compound. Most of the 4,6-disubstituted analogs were
inactive in reducing the viral titer. Importantly, the most potent
compound in this series is structurally similar to GNF-2 but is
devoid of c-Abl inhibitory activity.
[0275] In contrast to the 4,6-pyrimidine compound series,
2,4-disubstituted pyrimidine analogs yielded compounds with even
greater potency in the DENV yield reduction assay. In particular,
compounds with a trifluoromethoxy aniline at the C4 position of the
pyrimidine ring were found to be more potent than compounds
containing the aryl benzamide of the parent compound. Several
compounds in this series reduced viral yields to undetectable
levels under the conditions of these experiments. These data
suggested that in the 2,4-pyrimidine series, the ortho- or
meta-trifluoromethoxy substituted anilines at the C2 position were
a favorable chemical framework for obtaining compounds that
inhibited dengue virus potently.
[0276] A further focused SAR study was performed by preparing
analogs containing an ortho-trifluoromethoxy substituted aniline at
the 2-position of the 2,4-pyrimidine ring. As the 4-position of
2,4-dichloropyrimidine is more reactive than the 2-position,
various anilines and amines were first reacted with
2,4-dichloropyrimidine to prepare 4-substituted pyrimidines. The
mono-aminated products were then subjected to a second amination
with ortho-trifluoromethoxy aniline under acidic conditions to
obtain the final products. The compounds were tested for anti-DENV
activity in the viral yield assay under pre-/co-infection
conditions as described above. Viral yield was reduced by 100- to
1000-fold with pyrimidines bearing an aniline at the C2 position,
consistent with the previous series in which the most active
compounds contained 3 aromatic rings connected through an amide or
ether linkage (e.g., compounds X and Y). Compound 63 was the most
potent compound in this series, causing a 3 log unit drop in viral
titer at 75 .mu.M concentration of compound. To determine the
optimal position of the trifluoromethoxy group on the aniline
attached at the C2 position, we compared the most active compounds
with analogs in which the trifluoromethoxy aniline was moved to the
meta position (FIG. 5B). While aniline substitutents at the C4
position retained potent antiviral activity in the meta-series,
benzyl amine substituents at the C4 position lost activity in
comparison to the ortho-series where a robust 2-log unit drop in
viral titter was observed. For example, compounds 65 and 66 in the
meta series were less active than compounds 57 and 62 in the ortho
series. To explore substitutions at the C5 position of the
pyrimidine ring, we prepared a limited set of compounds (compounds
64 and 73-75, FIG. 5) and found that introduction of a chloride at
the C5 position was tolerated only in the ortho series and that
replacement of the pyrimidine core with a purine core resulted in a
complete loss of activity (data not shown).
Example 10
GNF-2 Acts at Two Separate Parts of the DENV Infectious Cycle
[0277] To examine the point(s) in the DENV infectious cycle
affected by GNF-2, we performed order of addition experiments
measuring the effect of GNF-2 when incubated with cells or virus
prior to infection (PRE), when added at the time of infection (CO),
or when added after the initial infection had been allowed to occur
(POST). To additionally probe the extent to which GNF-2's
anti-DENV2 activity may be mediated by Abl kinases, we performed
parallel experiments with imatinib, a well-characterized compound
with comparable activity against Abl kinases in biochemical and
cellular assays, but with a pharmacophore and molecular mechanism
distinct from GNF-2's. GNF-2, imatinib, or DMSO was present in the
viral inoculum and/or in the cell culture medium at various times
during the experiment. BHK21 cells were inoculated with DENV2 at a
multiplicity of infection (MOI) of 1 and incubated for one-hour at
37.degree. C. after which the inoculum was removed, the cells were
washed, and fresh medium was added to limit infection to a single
round. At twenty-four hours post-infection, corresponding to
approximately one complete life cycle of DENV, the yield of
infectious virions that had been released to the culture
supernatant was quantified by plaque-formation assay (PFA) as a
measure of successful DENV replication. We observed that both GNF-2
and imatinib inhibited DENV2 replication when added directly after
viral infection, and addition of either inhibitor five hours
post-infection caused a comparable level of inhibition. This
suggested that GNF-2 and imatinib affect events downstream of viral
entry since internalization and fusion of dengue virions has been
shown to occur on the order of minutes (average time 12.5 minutes)
following attachment to the cell surface. In subsequent
dose-response experiments, we determined that GNF-2 and imatinib
have comparable inhibitory potencies against this post-entry step,
with IC.sub.90 values--defined as the concentration of compound
sufficient to cause a 10-fold decrease in the single-cycle viral
yield--of 8 .mu.M observed for both compounds when added to cells
post-infection.
[0278] Somewhat unexpectedly, we also observed significant
inhibition of DENV2 when GNF-2 was pre-incubated with the viral
inoculum prior to infection but no inhibition of DENV2 when the
inoculum was pre-incubated with imatinib or when GNF-2 or imatinib
were added to cells at the time of inoculation. Together, these
observations suggested that this anti-DENV2 activity may be due to
a target present in the viral inoculum and be mediated by an
Abl-independent mechanism. To quantify this effect of GNF-2 on
DENV2 infectivity, we performed dose-response experiments in which
the virus inoculum was pre-incubated with varying concentrations of
compound at 37.degree. C. for 45 minutes prior to inoculation of
cells. The infection was permitted to proceed for one hour at
37.degree. C. followed by washes to remove non-adsorbed virus and
compound, replacement of fresh medium lacking compound, and then
quantification of viral yield at 24 hours post-infection by plaque
formation assay.
[0279] To further examine the idea that GNF-2 inhibits DENV2 via
two separate mechanisms, one mediated by Abl kinases at a
post-entry step and one mediated by an independent target
relatively early in the infectious cycle, we performed additivity
experiments using GNF-2, imatinib, and NITD6, a previously
validated inhibitor of DENV2 entry shown to interact with the DENV2
envelope protein and found to inhibit DENV2 infectivity in our
assay with an EC.sub.90 value of 200 nM. As expected, additive
inhibitory effects were observed when pre-treatment of the inoculum
with NITD6 was combined with treatment of DENV2-infected cells with
imatinib. These additive anti-DENV activities were recapitulated by
pre-incubation of the inoculum with GNF-2 and post-incubation of
DENV2-infected cells with GNF-2, suggesting that GNF-2 inhibits
DENV2 via two distinct mechanisms. Since GNF-2's activity as an Abl
kinase inhibitor has been well-documented, we next focused on
studying the target and mechanism responsible for its activity as
an inhibitor of DENV2 infectivity.
Example 11
GNF-2 Interacts Directly with the Dengue Virus Virion
[0280] Since GNF-2's effect on DENV2 infectivity required its
pre-incubation with the virus inoculum prior to infection, we
hypothesized that GNF-2 might target the dengue virion directly. To
explore this hypothesis, we synthesized a derivative in which the
exocyclic amide of GNF-2 is connected via a polyethylene glycol
linker to biotin. The resulting compound, biotin-GNF-2 was found to
inhibit DENV2 in the infectivity assay with an EC.sub.90 value
(.about.18 .mu.M), comparable to that of the parental GNF-2.
Additionally, the KD of GNF2-biotin for DENV2 soluble perfusion E
is around 1 .mu.M. See FIGS. 35-38.
[0281] Purified DENV2 virions were incubated with biotin-GNF-2 at
37.degree. for 45 minutes after which the reaction mixture was
mixed with streptavidin beads for affinity capture of biotin-GNF-2.
The streptavidin beads were washed ten times to remove
non-specifically bound material and then boiled in SDS buffer.
Western blot analysis of the SDS buffer eluate for the presence of
the DENV2 E protein demonstrated affinity capture of the DENV2 E
protein that was dependent on the presence of biotin-GNF-2.
Parallel negative control experiments performed with purified
particles of vesicular stomatitis virus (VSV), an enveloped,
negative-sense RNA virus, showed no capture of this unrelated virus
and demonstrated that the biotin-GNF-2-mediated affinity capture of
viral particles was specific for DENV2. Competition with compound
1-100-1 demonstrates that the interaction of virions with
biotin-GNF2 is reversible and that the association is not simply
aggregation.
[0282] This experiment demonstrates a direct interaction with
virions; the location of the interaction and the binding affinity
are investigated in Examples 12 and 13.
Example 12
Measurement of Binding Affinity Between GNF-2 and Dengue Virus
Envelope Protein by OCTET RED384
[0283] Using recombinantly expressed, soluble form of the prefusion
E dimer (sE.sub.2), we performed equilibrium affinity measurements.
Octet RED384 and streptavidin biosensors (from Fortebio) was used
for binding affinity studies. Streptavidin biosensor tips were
hydrated in loading buffer (1.times.PBS, pH 7.4 with, 2% DMSO) for
at least 10 min at room temperature. SA tips were saturated with
0.5 .mu.M biotinylated-GNF2 or DMSO (as control) for in 5-10 min
and then quenched by 10 .mu.g/mL Biocytin in 2 min. The biosensor
then were equilibrated in running buffer (1.times.PBS, pH 7.4 with,
2% DMSO, 0.1% BSA and 0.02% Tween 20) for 10 min before collecting
baseline in running buffer. Association of sE(wt) or sE(M196V)
mutant to biotinylated compounds were measured at minimum four
protein concentrations from 2 .mu.M to 30 .mu.M in 10-20 min and
dissociation were followed by dipping the biosensor into running
buffer in 10 min. The data were analyzed by ForteBio software for,
global fitting and steady state kinetics. KDs were obtained from
steady state kinetics analysis. See FIG. 42 and FIG. 43.
Example 13
Measurement of IC50 by Fluorescence Polarization Assay
[0284] The DV2 sE.sub.2(wt) dimer or corresponding DI/DII monomer
at different concentrations from 0 to 36 .mu.M were incubated with
40 nM FITC compound in kinetics buffer (1.times.PBS, pH 7.4 with,
2% DMSO) in low-volume 384 well microplates at RT for 2 hours or at
4.degree. C. for 24 hours. The fluorescence polarization
measurements were recorded in a PerkinElmer EnVisions instrument
(excitation wavelength, 485 nm; emission wavelength, 535 nm). The
FP data were analyzed by Origin9 and fitted by dose response
model.
Example 14
Selection of a Resistant Mutant
[0285] DENV2 NGC (MOI 0.1) was incubated with selected compounds
for 45 minutes at 37.degree. C. before addition to 10.sup.6 Vero
cells in a T25 flask. After a one hour infection, 5 mL medium was
added (2% FBS in DMEM) containing compound or DMSO. Infections
proceeded for four days, at which point supernatant was harvested
and spun briefly to remove cell debris. Five hundred .mu.L of
supernatant was used to infect a fresh T25 of Vero for one hour, at
which point 4.5 ml of medium containing compound was added. Viral
titer of each passage was determined by plaque-forming assay. The
first passaging with compounds maintained 20 .mu.M of each compound
over eight passages. The second passaging started at 2.5 .mu.M and
increased by 2.5 .mu.M each passage until 20 .mu.M was reached,
which was kept constant for two passages. Consensus sequencing of
the 7-148-6-resistant population identified mutation M196V in the E
protein, which maps to a beta-strand at the base of the
beta-octoglucoside binding pocket identified in the crystal
structure of the prefusion E dimer. See FIG. 39.
[0286] The M196V mutation was independently introduced to DENV2 E
in a single-cycle reporter virus system and confirmed that it
confers decreased sensitivity to 7-148-6. See FIG. 40.
[0287] Testing of the 7-148-6-resistant DENV2 quasispecies against
other disubstituted pyrimidine entry inhibitors suggested that it
confers decreased sensitivity to additional members of both the
2,4-disubstituted pyrimidine series (e.g., 2-12-2, 8-24-3) and the
4,6-disubstituted pyrimidine series (e.g., 1-100-1) when compared
to a DMSO-passaged control strain although the mutant appears to
still be sensitive to GNF-2. See FIG. 41.
EQUIVALENTS
[0288] While specific embodiments of the subject invention have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the invention will become apparent
to those skilled in the art upon review of this specification. The
appended claims are not intended to claim all such embodiments and
variations, and the full scope of the invention should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such
variations.
[0289] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference. In case of conflict, the present
application, including any definitions herein, will control.
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